Cretinism with combined hormone deficiency caused by a mutation in the PIT1 gene.

Cretinism is marked by irreversible mental and growth retardation. We describe here an entirely new case of cretinism showing combined pituitary hormone deficiencies of thyrotropin, growth hormone and prolactin that appears to be caused by homozygosity for a nonsense mutation in the gene for the pituitary specific transcription activator, Pit-1/GHF-1 (designated PIT1 in humans for pituitary specific factor 1). This is the first report in humans of a defect in a transcription activator causing deficiency of multiple target genes.

Relationship between proliferation and cell cycle-dependent Ca2+ influx induced by a combination of thyrotropin and insulin-like growth factor-I in rat thyroid cells.

The mechanism of cell proliferation by a combination of thyroid-stimulating hormone (TSH) and insulin-like growth factor-I (IGF-I) was studied in rat thyroid (FRTL-5) cells. IGF-I stimulated an approximately 3.5-fold increase in the rate of Ca2+ influx sustained for at least 6 h in TSH-pretreated cells but not in quiescent cells. The significant cell proliferation was observed when TSH-primed cells were incubated with IGF-I for 24 h but not for 12 h. IGF-I stimulated the rate of Ca2+ influx in a dose-dependent manner that was similar to that for induction of DNA synthesis. Both Ca2+ influx and DNA synthesis observed in response to IGF-I in TSH-primed cells were inhibited by cobalt. In addition, the stimulations of Ca2+ influx and DNA synthesis by IGF-I were dependent on extracellular Ca2+ in TSH-pretreated cells. When TSH-primed cells were pretreated with pertussis toxin, both IGF-I-induced Ca2+ influx and DNA synthesis were abolished. However, pertussis toxin did not block the priming action of TSH or forskolin. When calcium entry was induced by Bay K8644, it stimulated cell growth in TSH-primed cells but not in quiescent cells. Moreover, cobalt and lanthanum inhibited DNA synthesis even when added several hours after the addition of Bay K8644 but not when added 24 h after the growth factor in TSH-primed cells. These findings suggest that at least two important mechanisms may work in response to IGF-I only in the TSH-primed G1 phase of the cell cycle: first, IGF-I can activate directly or indirectly the Ca2+ channel via a pertussis toxin-sensitive substrate in TSH-primed cells; and second, a long lasting calcium entry by IGF-I may be a cell cycle-dependent mitogenic signal.

Lamellar body depletion in dogs undergoing pulmonary artery occlusion.

We have investigated the relationship between pulmonary artery occlusion (PAO) and the surfactant system of the lung by studying the ultrastructural responses of type II alveolar pneumocytes to PAO of 4-12 h duration in 16 mongrel dogs. In six of these animals, the occluded lung was allowed to reperfuse for 6 h before killing and in four animals subjected to PAO of 4 h duration, the occluded lung was ventilated with 5% CO2 balance air. PAO by itself resulted in a dramatic 80% reduction in the volumetric density of lamellar bodies (LB) in the type II cells. This resulted predominantly from a decrese in volume of the individual LB. Although reperfusion was associated with an increase in LB volume density toward normal, 6 h of reperfusion was insufficient to re-establish normal type II cellular morphology. Ventilation of the occluded lung with 5% CO2 prevented LB depletion indicating that alveolar CO2 tension may affect the release and/or synthesis of LB in type II pneumocytes.

Evolutionary molecular engineering by random elongation mutagenesis.

We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/1222T)-the thermostability of which is much lower than that of the wild type-was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.

Symptomatic colonic spirochaetosis in an immunocompetent patient.

Spirochaetes are organisms that can infect the colon of people with normal or compromised immune systems. Infected patients can present with a variety of gastrointestinal symptoms, including diarrhoea and rectal bleeding. However, some report a lack of association between specific symptoms and the presence of spirochaetes. It is therefore unclear whether the spirochaetes colonising the colon are true pathogens. Diagnosis is typically made by histological examination, with the biopsy specimen showing a band-like growth of spirochaetes adherent to the colonic luminal surface, giving an accentuated brush-border appearance. A course of metronidazole can eliminate the spirochaetes, but treatment might not lead to improvement of symptoms. Owing to the lack of a definite association between symptoms and the presence of spirochaetes, observation without specific antibiotic treatment can be pursued in most patients.

Clonal isolation of epithelial cells from mouse lung adenoma.

Clones of epithelial-like cells were established from urethan-induced mouse lung adenoma. Electron microscopy of one clone showed that the cells contained lamellar inclusion bodies similar in appearance to those seen in the adenoma precursor, the type II alveolar pneumocyte. The clones exhibited characteristics associated with both "transformed" and "normal" cells in culture; i.e., although aneuploid, the cells grew at a slower rate than most transformed cells, did not form colonies in soft agar and, after prolonged subculture, were not tumorigenic when transplanted s.c. into appropriate hosts. Hydrocortisone treatment of the cloned cells led to growth stimulation and the eventual acquisition of neoplastic potential. Epithelial tumors were produced more readily in athymic, nude mice than in antilymphocyte serum-treated A/He mice. The cells are producing a C-type RNA virus into the culture medium.

Nucleotide sequence of the complementary DNA for human Pit-1/GHF-1.

Human cDNA clones encoding Pit-1/GHF-1, a pituitary-specific DNA binding factor, were obtained by PCR following reverse transcription of human pituitary RNA. It is approx. 1.3 kb in size with 0.1 kb 5' non-coding region, 0.9 kb protein-coding region and 0.3 kb 3' non-coding region. The predicted human Pit-1/GHF-1 peptide structure has 291 amino acids and is highly conserved among mouse, rat and bovine. In addition, the 5' non-coding region is highly conserved with rat pit-1/GHF-1 sequence to the transcription start site.

Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver.

We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 +/- 2 mg/h per 100 g liver, hemopexin at 5.0 +/- 0.7, haptoglobin at 3.2 +/- 0.6 and transferrin at 5.1 +/- 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 +/- 0.1, and that of lactate: pyruvate 27 +/- 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies. A minimal effect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3-4-fold increase in hemopexin but not in albumin and transferrin synthesis. A striking 2-3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.

The distinct roles of alpha- and beta-subunits of human thyrotropin in the receptor-binding and postreceptor events.

The roles of alpha- and beta-subunits of human TSH (hTSH) were studied by investigating the inhibitory effects of monoclonal antibodies on the biological activity of hTSH. Four monoclonal antibodies directed toward different epitopes on the beta-subunit (hTSH beta) inhibited completely the receptor binding of hTSH to FRTL-5 rat thyroid cells. In contrast, five monoclonal antibodies directed toward different epitopes on the alpha-subunit had little or no effect on the receptor binding. Furthermore, four of five alpha-subunit-specific antibodies and three of four hTSH-specific antibodies inhibited both hTSH-induced cAMP accumulation and thymidine uptake in FRTL-5 cells, in a dose-response manner. One hTSH (alpha-beta heterodimer)-specific antibody which did not recognize the free subunits also had the inhibitory effect on both the receptor binding and hTSH-induced cAMP accumulation. These results strongly suggest that hTSH beta is indispensable for recognizing the receptors on FRTL-5 cells and that the alpha-subunit is required for signal transduction in postreceptor step(s). In addition, it is also suggested that the highly conformational structure of hTSH is absolutely essential for the biological activity.

Effect of clomiphene on basal and LRH-induced gonadotropin secretion in postmenopausal women.

LRH tests were performed in 11 postmenopausal women before and after the administration of 200 mg of clomiphene citrate daily by mouth for 7 consecutive days. The basal levels and the maximum increments of serum LH (deltaLH) and the area under the response curve from basal level (deltaarea) after LRH administration, significantly (P less than 0.005) decreased after consecutive administration of this compound. Serum FSH levels were significantly (P less than 0.025) decreased but deltaFSH and deltaarea in LRH test were statistically unchanged. These results suggest that clomiphene citrate in postmenopausal women inhibits the pituitary gonadotropin response to exogenous LRH by its estrogenic effects.

Exophthalmos in autoimmune thyroid disease.

Protrusion of the eyes of patients with autoimmune thyroid disease was compared with that of healthy subjects. The mean values for protrusion in patients with thyrotoxic Graves' disease and Hashimoto's disease and in healthy subjects were 16.6 +/- 2.1 mm (mean +/- SD; n = 122), 14.2 +/- 1.8 mm (n = 100), and 13.9 +/- 1.9 mm (n = 558), respectively. The value of Graves' disease was significantly different from that for healthy subjects (P < 0.001). Individual values for protrusion showed a similar normal distribution in these three groups, but were displaced to higher values as a whole in Graves' disease. These results, which suggest that almost all patients with Graves' disease have exophthalmos, do not support the idea that Graves' ophthalmopathy is a distinct single autoimmune disease.

Comparison of short and long-term treatment with synthetic LH-releasing hormone and clomiphene citrate in male hypothalamic hypogonadism.

The effects of short and long-term treatment with clomiphene citrate and with synthetic LH-releasing hormone (LHRH) on gonadotropin release were studied in 5 male patients with hypothalamic hypogonadism. Neither short-term treatment with clomiphene citrate (50 mg or 200 mg daily for 4 days) nor long-term treatment (25 mg or 100 mg for 30-62 days) was effective in increasing serum LH and FSH in any patients. On the other hand, 5 cases showed slight or no rise in LH after administration of a single dose of 100 mug LH-releasing hormone (LHRH) and higher rises after administration of 400 mug. Four cases who were given 200 mug LHRH daily for 3 weeks showed an increasing response of serum LH and reached maximal LH levels at 2 weeks, followed by no further increase. In two of the four cases serum LH level reached normal adult male ranges. There was no increase of serum testosterone during LHRH treatment. These results suggest that long-term treatment with synthetic LHRH may be effective for gonadotropin restoration in some patients with hypothalamic hypogonadism in which long-term treatment of clomiphene citrate was shown to be ineffective.

LH and FSH response to synthetic LHRH after consecutive administration of clomiphene citrate in normal males.

LHRH tests (100 mug iv) were performed in 11 normal male volunteers before and after administration of 100 mg clomiphene citrate per os for 7 days. Serum FSH, both pre and post LHRH levels (peak value and area under response curve) were significantly increased (P less than 0.01) by clomiphene citrate, while the maximum increments of FSH above baseline (deltaFSH) and area under response curve from basal level (deltaarea) were unchanged. On the other hand, basal serum LH levels increased significantly (P less than 0.005), post LHRH levels did not change by clomiphene citrate, whereas the increments after LHRH administration (both deltaLH and deltaarea) decreased significantly (P less than 0.05) on the second LHRH test. Basal levels of testosterone increased following clomiphene citrate treatment (P less than 0.005). The increased testosterone is considered to suppress the effect of LHRH on LH secretion.

Effect of prolonged LH-releasing hormone administration on gonadotropin response in patients with hypothalamic and pituitary tumors.

The effect of prolonged administration of synthetic LH-RH (400 mug daily im for 5 days) on gonadotropin response to LH-RH (100 mug single injection iv) was compared in 13 patients with pituitary tumors or suprasellar tumors. No patients with pituitary tumors exhibited an augmented response on serum FSH and LH after prolonged LH-RH administration. A paradoxical fall in response to LH-RH test occurred in 3 patients with pituitary tumors and in a patient with hypothalamic tumor in whom gonadotropin responses were normal on the first LH-RH test. On the other hand, 2 patients with suprasellar tumors with a low response to the initial LH-RH test showed significant increase on a second LH-RHtest after consecutive LH-RH administration. Consecutive administration of LH-RH may help to distinguish between the hypogonadism of pituitary origin and that of hypothalamic origin when the initial LH response to a single dose of LH-RH is subnormal.

Serum thyroid hormones and thyrotropin in anorexia nervosa.

Sixteen patients with typical signs and symptoms of anorexia nervosa were studied with measurement of serum thyroxine (T4), triiodothyronine (T3) and thyrotropin (TSH), both baseline and stimulated by thyrotropin-releasing hormone (TRH). The results of the patients were compared with those of 16 normal control subjects. Serum T4 (5.8 plus or minus 0.26 mug/100 ml, mean plus or minus SE) and T3 (82 plus or minus 5.7 ng/100 ml) of patients with anorexia nervosa were significantly (P less than 0.001) lower than those of control subjects (T4 7.7 plus or minus 0.32 mug/100 ml and T3 158 plus or minus 4.7 ng/100 ml respectively). Furthermore, the ratio of T3/T4 (1.48 plus or minus 0.243 x 10(-2)) in anorexia nervosa was also lower than that of control subjects (2.21 plus or minus 0.093 x 10(-2)) (P less than 0.001). Basal serum TSH was within normal or below the limits of detection. TSH and T3 rose after administration of TRH. The peak values of TSH were observed after 60 to 12o min, instead of 30 min normally seen after TRH injection.

Effect of triiodothyronine treatment on prolactin secretion in patients with amenorrhea-galactorrhea.

In order to determine the role of thyroid hormone in prolactin (PRL) secretion in patients with amenorrhea-galactorrhae, PRL response to 500 mug of iv thyrotropin-releasing hormone (TRH) was studied before and after the administration of triiodothyronine (T3) in 10 patients with amenorrhea-galactorrhea. Seven of these patients were euthyroid and the other 3 had hypothyroidism. The patients in the euthyroid group received 50 mug of T3 daily for 7 days and 75 mug q.d. for the ensuing 14 days. The hypothyroid patients received T3 at progressively increasing doses from 10 mug q.d. to 75 mug q.d. during 34 to 68 days. In the initial test, the elevated basal levels of PRL, 61.9 +/- 9.8 ng/ml (Mean +/- SE) exhibited a slight but insignificant net increase (7.7 +/- 2.1 ng/ml) after TRH injection in the euthyroid group. However, a marked response to TRH with a net increase of 147.2 +/- 26.3 ng/ml from the basal level of 47.3 +/- 11.2 ng/ml was observed in the hypothyroid patients. After treatment with T3, both the basal level (56.9 +/- 8.3 ng/ml) and the net increase (9.9 +/- 3.6 ng/ml) of PRL following TRH stimulation remained virtually unchanged in the euthyroid group. The hypothyroid group, in contrast, displayed a significant depression of both the basal level (26.1 +/- 13.0 ng/ml) and the net increase (33.8 +/- 6.5 ng/ml) of PRL to TRH stimulation. The diminution of the basal levels and responses of thyroid-stimulating hormone (TSH) to TRH stimulation was observed in all cases of both groups. These results suggest that the level of thyroid hormone has little pathogenic role in PRL secretion in euthyroid patients with amenorrhea-galactorrhea, in contrast to its marked effect in hypothyroid patients with amenorrhea-galactorrhea.

Transient recurrence of hyperthyroidism after delivery in Graves' disease.

Four patients with Graves' disease whose hyperthyroidism was in remission following antithyroid therapy were studied without any treatment during and after pregnancy. In the 8-9th month of pregnancy, they were in a euthyroid state with serum levels of thyroxine (T4) of 18.9, 11.9, 11.2 and 14.5 mug/100 ml, triiodothyronine (T3) of 273, 190, 162 and 244 ng/100 ml and T3 resin sponge uptake (RT3U) of 22, 14, 19 and 16% respectively (normal pregnant range: T4, 7.0-15.0, T3 140-250, RT3U 15-25). At 1-3 months after delivery, hyperthyroidism recurred, as manifested by T4 levels of 17.2, 14.5, 16.7 and 21.7 mug/100 ml, T3 levels of 320, 225, 390 and 464 ng/100 ml and RT3U levels of 34, 34, 43, and 41% respectively (normal non-pregnant range: T4 5.0-12.0, T3 90-190, RT3U 24-37). The recurrence of hyperthyroidism was also demonstrated by serial measurements of serum free T4 and free T3. The thyroid function of all four patients returned spontaneously to the normal range at 4-6 months after delivery. One patient developed hypothyroidism for a short period before regaining the euthyroid state. The titers of serum anti-thyroid microsomal antibodies and levels of serum immunoglobulins decreased during pregnancy and increased transitorily at the time of hyperthyroidism after delivery. Similarly, increases in the levels of thyroid hormones, anti-thyroid antibodies and immunoglobulins were observed transitorily following spontaneous abortion after 4 months' pregnancy in one case. We suggest that the transient recurrence of hyperthyroidism in Graves' disease may be induced by immunological changes after delivery.

Pituitary-thyroid function in trophoblastic disease.

Pituitary-thyroid function was assessed in 12 patients with trophoblastic disease (4 hydatidiform mole, 3 invasive mole, and 5 choriocarcinoma). Thyroid-stimulating activity was detectable, by means of the McKenzie bioassay, in 6 patients (Group 1) but not in the other 6 patients (Group 2). In Group 1 serum thyrotropin (TSH) determined by radioimmunoassay was mostly undetectable and did not respond to the administration of thyrotropin-releasing hormone (TRH) determined by radioimmunoassay was mostly undetectable and did not respond to the administration of thyrotropin-releasing hormone (TRH), while in Group 2 basal TSH was detectable in half of the patients and responded to TRH in all cases. Serum concentrations of total thyroxine (T4) (18.7 +/- 2.0 mug/100 ml, mean +/- SE), free T4 (4.9 +/- 0.04 ng/100 ml), total triiodothyronine (T3) (352 +/- 72 ng/100 ml), and free T3 (0.57 +/- 0.11 ng/100 ml) in Group 1 were statistically greater than those in Group 2 (total T4, 9.2 +/- 1.0 mug/100 ml, free T4, 2.0 +/- 0.2 ng/100 ml; total T3 156 +/- 20 ng/100 ml, and free T3 0.23 +/- 0.03 ng/100 ml). Free T4 and T3 fractions were within normal limits in both groups. After treatment of 5 patients in Group 1, the thyroid stimulating activity determined by bioassay dropped to undetectable levels, the serum concentrations of thyroid hormones decreased to normal limits, and TSH response to TRH became positive. These findings indicate that an abnormal thyroid stimulator, derived from the trophoblastic tissue, stimulated the thyroid hormone secretion from the thyroid gland and in turn suppressed TSH response to TRH in some patients with trophoblastic disease.