Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Changes in plasma fibronectin levels in thyroid diseases

The plasma levels of fibronectin (Fn) have been measured in normal subjects and in patients with thyroid diseases. The mean plasma Fn levels in 62 normal adults was 32.0 +/- 6.0 mg/dl, whereas it was elevated to 62.6 +/- 16.1 mg/dl (mean +/- SD) in 25 patients with hyperthyroidism and decreased to 19.2 +/- 8.0 mg/dl in 9 patients with hypothyroidism. The 9 patients with simple goiter have normal values of 29.1 +/- 8.0 mg/dl. With the administration of anti-thyroid drugs, plasma Fn levels normalized, with a time lag, in parallel with normalization of the thyroid function. Positive correlation was obtained between Fn levels and serum levels of triiodothyronine (T3) and thyroxine (T4). The present findings indicate that measurement of plasma Fn both in the basal state and during treatment provides evidence of altered Fn metabolism in thyroid diseases and serves to follow up the effect of treatment.     

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

High and testosterone-dependent glucose 6-phosphatase activity in epithelium of mouse seminal vesicle

For study of the mechanism of seminal fructogenesis, glucose 6-phosphatase activity was examined cytochemically (a method modified from that of Wachstein and Meisel) and biochemically (the method of Leskes et al.) in seminal vesicles from normal, castrated, and castrated and testosterone-treated mice. The reaction product for the activity was localized in the endoplasmic reticulum and nuclear envelope of all cell types composing the seminal vesicle. In normal seminal vesicle, the reaction product was apparently more abundant in columnar and basal cells than in other cell types. Ten, 20, and 30 days after castration, the abundant amount of reaction product in columnar and basal cells decreased to the level in other cell types. In animals treated with testosterone after castration, however, the reaction product in columnar and basal cells remained abundant. If fructose 6-phosphate was added to the reaction medium in place of glucose 6-phosphate, the amount and pattern of deposition of the reaction product did not change. Changes in biochemical activity in castrated or castrated and testosterone-treated animals paralleled the cytochemical results. The results show that the high activity in columnar and basal cells is under the control of testosterone, and the role of this enzyme is probably to release fructose into the seminal fluid.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Effects of N-fertilizers,straw, and dry fallow on the nitrogen balance of a flooded soil planted with rice

Summary Nitrogen balance studies were made on rice (Oryza sativa) grown in flooded soil in pots. A low rate of fertilizer (5.64 mg N. kg⁻¹ soil) did not depress the N gain, but a high rate (99.72 mg N. kg⁻¹ soil) elminated the N gain. Soil N loss was negligible since¹⁵N applied as ammonium sulfate and thoroughly mixed with the soil was recovered from the soil-plant system after 3 crops. The observed N gain, therefore, was caused by N₂-fixation, not by a reduction of soil N loss. Straw enhanced N gain at the rate of 2–4 mg per g straw. However, this gain was not observed when soil N availability was high. Dry fallow between rice crops decreased the N gain.     

Structure of the trypsin-binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.