Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification.

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.


Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney by two-step affinity chromatography on fetuin and heparin columns and subsequent anion-exchange high-performance liquid chromatography. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified fraction gave two protein bands corresponding to proteins of relative molecular mass 33,000 (p33) and 41,000 (p41), respectively. Although the proteins had no haemogglutinating activities towards human and rabbit erythrocytes, their carbohydrate-binding activity was examined by a newly developed method using horseradish peroxidase (HRP) and/or biotin-labelled glycoconjugates as affinity probes. They could bind in a Ca(2+)-dependent manner to labelled fetuin and heparin in a specific and dose-dependent manner by solid-phase assay after immobilization on plastic plate surface. Inhibition assay of the binding revealed that N-acetylneuraminic acid is the most potent inhibitor of the proteins among the monosaccharides tested. Fucoidin and heparan sulphate most strongly inhibited the binding of the proteins to labelled heparin. Direct binding assay to acidic glycolipids prepared from bovine kidney showed that the proteins react with the ganglioside fraction but not with sulphatide [Gal(3-SO4) beta 1-1Cer]. These results indicated that the purified proteins have a significant affinity to charged oligosaccharides linking to glycoproteins, glycolipids and charged polysaccharides in a Ca(2+)-dependent manner.


Affinity chromatography in purification of A1 adenosine receptors.

Purification of A1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A1 adenosine receptor was solubilized with digitonin-cholate from brain membranes and then purified by a sequential use of affinity chromatography on XAC-agarose, hydroxyapatite chromatography and reaffinity chromatography on XAC-agarose. The A1 adenosine receptor was purified ca. 45,000-fold with a yield of 5%. The final receptor preparation gave a single broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr approximately 34,000. This band was also shown to be specifically labelled with an affinity labelling reagent for A1 adenosine receptors. This purification method was also applicable for the complete purification of A1 adenosine receptors from rat testis and human brain membranes.


Characterization of the interaction between human plasma fibronectin and collagen by means of affinity electrophoresis.

The interaction between human plasma fibronectin and different types and forms of collagen were analysed by affinity electrophoresis at different pH values. The fibronectin bound tightly to collagen type I, III and IV, but not to type V. The fibronectin interacted better with the denatured form of collagen type I (gelatin) than with the native form. At pH less than 5.5 the fibronectin exhibited much lower affinity to gelatin than at pH greater than 8.0. The interaction between the fibronectin and gelatin was further analysed by affinity electrophoresis in which apparent dissociation constants (Kd) of the fibronectin for gelatin were calculated, and effects of urea, 2-mercaptoethanol and temperature on the interaction were examined. The fibronectin markedly diminished its affinity to gelatin at 3 M urea to give Kd = 2.5 x 10(-6) M, which was 1000 times larger than the value without urea. The fibronectin dissociated into its monomers and the monomers diminished their affinity to gelatin in a stepwise fashion with increase in concentration of 2-mercaptoethanol. The fibronectin diminished the affinity to gelatin by elevating temperature, and van't Hoff plots of log Kd values against the reciprocal of absolute temperature (T) showed that log Kd was inversely proportional to 1/T in the range 15-50 degrees C, and the thermodynamic parameters of the standard enthalpy change, the standard free energy change and the entropy change at 37 degrees C for association of fibronectin and gelatin were all negative. At 60 degrees C the affinity of fibronectin to gelatin was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)


Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.


Separation of monoclonal antibodies from antihapten antisera by two-dimensional affinity electrophoresis.

A high-resolution two-dimensional affinity electrophoresis (2D-AEP) method was developed, using capillary polyacrylamide gel (PAG) isoelectric focusing in the first and slab PAG affinity electrophoresis in the second direction. Using this method, anti-hapten antibodies were separated into a number of monoclonal antibody [immunoglobulin G (IgG)] families, each of which is composed of several IgG spots having an identical affinity to the hapten but different isoelectric points. 2D-AEP may offer a powerful tool for solving fundamental problems in immunochemistry such as antibody heterogeneity, its hapten binding specificity and antigen-dependent somatic mutation.


Determination of the association constant of monovalent mode protein-sugar interaction by capillary zone electrophoresis.

Protein-sugar interaction was observed by capillary zone electrophoresis, using a few beta-galactose-specific lectins and lactobionic acid as protein and sugar models, respectively. The lectin peaks were retarded in a concentration-dependent manner by addition of lactobionic acid in a carrier, and association constants of monovalent mode interactions could be obtained from t1 (migration time of protein), t2 (migration time of complex, obtainable as the migration time at the plateau) and the slope of the (t-t1)-1 vs. [S]-1 plots, where t and [S] are the migration of protein in the presence of lactobionic acid and the concentration of lactobionic acid, respectively. The values for Ricinus communis agglutinin, peanut agglutinin and soy bean agglutinin at pH 6.8 were 3.3 . 10(3), 9.1 . 10(2) and 1.1 . 10(2)1 mol-1, respectively. This method required only small amounts of protein samples and was reproducible. The amount of the sugar could be minimized under the conditions that the carrier was a buffer containing the sugar whereas the electrode solutions consisted only of the buffer.


Process monitoring by flow-injection immunoassay. Evaluation of a sequential competitive binding assay.

A new variation on the theme of flow-injection binding assays is presented, namely the sequential competitive binding assay, in which the sample containing the native antigen is first introduced into the flow system followed by a pulse of labelled antigen. The flow-injection binding assay was used in monitoring the effluent from column chromatographic separations of proteins. By applying a computer-based evaluation system, concentrations are automatically read and compensation is made for denaturation in the affinity sorbent.


Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.


Occurrence of an allosteric transition in the modification of papain with L-1-acetyl-2,3-dihydropyrrolo[2,3-b]-indole-2-carboxamide.

When papain was reacted with L-1-acetyl-2,3-dihydropyrrolo[2,3-b]indole- 2-carboxamide at pH 8.0, inactivation occurred accompanied by modification of Cys-25 in the active site. Plots of pseudo first-order rate constants against the reagent concentrations yielded an anomalous sigmoidal curve, suggesting that papain responded to this reagent in an allosteric manner. This is supported by the fact that the presence of a moderate concentration (a twenty-fold molar excess) of N alpha-acetyl-L-tryptophanamide over papain accelerated the inactivation.


Specific assay for endotoxin using immobilized histidine, Limulus amoebocyte lysate and a chromogenic substrate.

The Limulus amoebocyte lysate (LAL) test is inhibited or enhanced by many substances. In order to overcome this problem, a specific endotoxin assay method using a membrane filter unit, a chromogenic LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins) was developed. Endotoxins are quantitatively adsorbed on immobilized histidine. The adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the membrane filter unit, and their activities are directly assayed with the LAL reagent in a filter cup without any inhibition or enhancement. The reproducibility and the accuracy of this method are high. This new endotoxin assay method using immobilized histidine can be used for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics, as an alternative to the more common gel-clot technique.


Use of various types of column reactors for flow-injection analysis.

Two or three different kinds of immobilized enzymes can be aligned in a minireactor so that sequential enzymatic reactions are carried out from upstream to downstream during flow-injection analysis. A lactate oxidase-catalase reactor, used as precolumn for removing pre-existing lactate in serum before the lactose dehydrogenase (LDH) reactions, was useful for the determination of serum LDH activity, which did not require any blank correction. A sequential glutamate dehydrogenase-glutamate oxidase reactor was also useful for a novel chemiluminometric determination of ammonia. On the other hand, a co-immobilized creatininase-creatinase-sarcosine oxidase reactor, in spite of containing creatininase which catalyses the reversible reaction, was the most efficient for the determination of serum creatinine.


Study of high-affinity interactions by quantitative affinity chromatography. Analytical expressions in terms of total ligand concentration.

Analytical expressions are derived for the description of ligand-facilitated and ligand-retarded desorption of partitioning solute in terms of total ligand concentration in quantitative affinity chromatography. Their application is then illustrated by consideration of results from recycling partition equilibrium studies of the heparin-facilitated desorption of thrombin from heparin-Sepharose, and of the competition between methyl-alpha-D-mannoside and p-nitrophenyl-alpha-D- mannoside for concanavalin A immobilized on CPG-170. Finally, published frontal affinity chromatographic data for the NADH-dependent elution of rabbit muscle lactate dehydrogenase from oxamate-Sepharose are reanalysed using these equations to demonstrate the characterization of a system reflecting the binding of a solute-ligand complex to an affinity matrix. This investigation extends the scope of quantitative affinity chromatography to include not only the study of solute-ligand interactions governed by larger binding constants but also the characterization of interactions in which the partitioning solute and ligand are both macromolecular, and eliminates the need for prior dialysis to establish the free ligand concentration required for application of earlier analytical expressions.


Recent developments in quantitative affinity chromatography.

This review surveys developments during the past decade in the use of quantitative affinity chromatography as a means of evaluating equilibrium constants for solute-ligand and solute-matrix interactions. Topics include allowance for multivalency of the partitioning solute, removal of the myth that highly substituted affinity matrices are unsuitable for zonal quantitative affinity chromatography, adaptation of the technique to allow characterization of high-affinity interactions and the application of quantitative affinity chromatography theory to the characterization of biospecific adsorption phenomena in cellular systems.


Base-atom recognition in protein adsorption to alkyl agaroses.

The three proteins phosphorylase b, calmodulin and fibrinogen are adsorbed onto thioalkyl derivatives of Sepharose much more strongly than onto gels carrying the same alkyl residue coupled via a carbamate linkage. This enhancement of binding onto alkyl-S-Sepharoses compared with alkyl-N-agaroses is not primarily due to an increase in the extent of conformational changes of the proteins occurring on the gel surface. This can be shown in experiments with the tripeptide Trp-Trp-Trp. The Trp tripeptide is also adsorbed with a much higher affinity to butyl-S-Sepharose than to butyl-N-Sepharose, showing that the primary interaction between the immobilized alkyl residue and the amino acids of the protein is decisive for adsorption. A model stressing the strong influence of an atom or a group of atoms at the base of an immobilized alkyl residue is described as "base-atom recognition".


Determination of maleic hydrazide residues in cured tobacco by gas chromatography.

A rapid and sensitive method for measuring maleic hydrazide (6-hydroxy-2H-pyridazin-3-one) residues in cured tobacco is described. A mixture of free and bound maleic hydrazide is extracted with hydrochloric acid in which maleic hydrazide glycoside is simultaneously hydrolysed. The free maleic hydrazide obtained is methylated using dimethyl sulphate and the derivative is partitioned into chloroform and determined by capillary gas chromatography using a nitrogen-phosphorus detector. The limit of detection of maleic hydrazide is 5 ppm.


Separation of non-steroidal anti-inflammatory agents using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) was investigated for the separation of non-steroidal anti-inflammatory agents (NSAIs). Three different stationary phases (SB-methyl-100, SB-biphenyl-30, and SB-cyanopropyl-50) were compared for the separation of the compounds. Baseline separation of a flufenamic acid, mefenamic acid, fenbufen and indomethacin mixture was achieved on the SB-biphenyl-30 column using a pressure gradient. A mixture containing flufenamic acid, mefenamic acid, acetylsalicylic acid, ketoprofen and fenbufen and another mixture containing ibuprofen, fenoprofen, naproxen, ketoprofen and tolmetin were well separated on the SB-cyanopropyl-50 column using pressure gradients. Typical analysis time for a mixture of NSAIs on the biphenyl or cyanopropyl column was approximately 20-25 min. Application of the method using the biphenyl column to the determination of NSAIs present in selected commercial dosage forms was demonstrated.


Active respiratory syncytial virus purified by ion-exchange chromatography: characterization of binding and elution requirements.

Two viruses, respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) were used to evaluate viral purification by an affinity resin column (Matrex Cellufine Sulfate (MCS); Amicon Division, WR Grace & Co.). Viable RSV was purified significantly from crude cell lysate by a single pass through a column containing the anionic MCS resin. Most cell protein and albumin eluted from the MCS resin with phosphate buffered saline (PBS) but RSV eluted at high ionic strength, i.e., greater than or equal to 0.6 M NaCl. Further purification was possible by sucrose step gradient centrifugation. The RSV prepared by column purification or by column plus sucrose gradient separation was both intact and infective. RSV and pure samples of VSV were used to optimize ionic strength and salts for elution from the MCS column: 0.8 M NaCl removed most of the viral protein. The capacity of the MCS gel for RSV or VSV was found to be about 0.6-0.8 mg viral protein per ml of hydrated resin. Detergent-solubilized viral membrane proteins bound to the MCS resin in 0.145 M NaCl and eluted with higher salt concentrations. Thus, this resin also may be a useful aid for relatively gentle purification of these proteins.


Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7.

A sandwich enzyme-linked immunsorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the nine different AHSV serotypes.


Progressive trabecular osteopenia in women with hyperprolactinemic amenorrhea.

Reductions in cortical and trabecular bone mass have been documented in young women with hyperprolactinemic amenorrhea. It is unknown whether trabecular osteopenia is progressive or reversible with treatment of hyperprolactinemia. In addition, it is not known whether clinical or hormonal variables can predict trabecular bone density (BD) changes. Therefore, we investigated prospectively trabecular BD by computed tomography in 52 hyperprolactinemic women and 41 controls. The mean follow-up interval was 1.8 +/- 0.1 (SEM) yr. Patient groups were defined as follows: group 1, amenorrhea during the entire study; group 2, restoration of menses during the study by treatment of hyperprolactinemia; group 3, regular menses despite hyperprolactinemia, with no history of prior amenorrhea; group 4, history of prior amenorrhea, but menses restored with treatment of hyperprolactinemia before study entry; and group 5, oligomenorrhea. Groups 1, 2, and 4 had significant (P = 0.0006) initial spinal osteopenia [mean BD 141 +/- 7 (SEM), 144 +/- 9, and 151 +/- 5 mg/cc K2HPO4, respectively] compared with controls or with group 3 (170 +/- 4 and 173 +/- 8 mg/cc K2HPO4, respectively). Group 5 had an initial mean BD which was midway between that of the amenorrheic and eumenorrheic women (156 +/- 13 mg/cc K2HPO4). Group 1 had a significant (P = 0.04) decrease in mean BD to 132 +/- 8 mg/cc K2HPO4 over 1.7 +/- 0.2 yr, with BD in 42% of the group more than 2 SD below the control mean at the final study point. The mean BD in group 2 increased to 155 +/- 9 mg/cc K2HPO4, approaching significance (P = 0.07) when compared with the initial BD. Five of the nine patients in this group (56%) had an increase in BD greater than the variation expected for the computed tomography technique. However, 44% of the group 2 patients had a spinal BD which remained more than 1 SD below the normal mean. There was no change in BD in the other groups.(ABSTRACT TRUNCATED AT 400 WORDS)


A longitudinal analysis of premenopausal bone loss in healthy women and women with hyperprolactinemia.

In this report we describe longitudinal measurements of forearm and spinal bone mineral in healthy women and women with hyperprolactinemia. One hundred and ten women underwent yearly assessment of forearm and spinal bone mineral by single photon absorptiometry and computed tomography for an average of 4.7 yr. At entry into the study, women with hyperprolactinemic amenorrhea had 21% lower spinal bone mineral and 2.5% lower forearm bone mineral than healthy premenopausal women. Despite decreased estradiol levels (31 +/- 23 pmol/L), spinal bone in women with hyperprolactinemic amenorrhea did not change over time (+0.08%/yr; P = 0.89). In contrast, spinal bone in healthy women with regular menses (mean age, 34.6 +/- 6.6 yr) decreased significantly (1.7%/yr; P = 0.01). Cortical bone in the forearm did not change in either group. The hyperprolactinemic subjects had higher body mass index (28 +/- 6 vs. 24 +/- 4 kg/m2) and serum testosterone (0.5 +/- 0.2 vs. 0.39 +/- 0.16 pmol/L) than control subjects, but neither parameter correlated with bone loss. Although 64% of the hyperprolactinemic subjects had serum estradiol levels below 30 pmol/L, there was no correlation between estradiol or duration of amenorrhea and bone loss. Women with normal PRL levels and regular menses 3-9 yr after treatment of hyperprolactinemia had significantly lower spinal bone mineral (147 +/- 28 mg/mL) than healthy premenopausal women (169 +/- 29 mg/mL) and showed no change in spinal bone (+0.3%/yr; P = 0.67) over 5 yr. Despite significant hypoestrogenemia, women with hyperprolactinemic amenorrhea did not evidence a rapid decline in spinal bone. Restoration of gonadal function was not associated with normalization of bone mineral. The bone loss that accompanies hyperprolactinemia is not comparable to that which occurs after oophorectomy or menopause. These findings raise important questions about the importance of osteopenia as an indication for treatment of hyperprolactinemic amenorrhea.


Attenuation of luteinizing hormone secretory burst amplitude as a proximate basis for the hypoandrogenism of healthy aging in men.

To evaluate the impact of healthy aging on specific features of endogeneous LH secretion and clearance, we applied deconvolution analysis to 24-h serum immunoradiometric LH concentration series obtained in normal men whose ages ranged from 21-73 yr. Deconvolution analysis was employed to quantitate the number, amplitude, duration, and mass of individual LH secretory bursts underlying the serum LH concentration profiles, and simultaneously estimate the half-life of LH in individual men. Plasma total and free testosterone and estradiol concentrations and body mass index (a measure of relative adiposity) were studied as possible significant covariates of age and LH secretion. We found that age was a negative determinant of LH secretory burst amplitude (r = -0.519, P = 0.013), and a positive predictor of LH secretory burst frequency (r = +0.435, P = 0.043) and basal LH secretory rates (r = +0.486, P = 0.029). Increasing age also correlated positively with LH secretory burst half-duration (duration of the secretory event at half-maximal amplitude, r = +0.656, P less than 0.001). In contrast, age did not relate to daily pulsatile LH production rate, the mass of LH secreted per burst, or the mean (24-h) serum concentration of immunoradiometric LH. Age correlated negatively with serum free testosterone (r = -0.622, P = 0.0034) but not estradiol concentrations. The serum free testosterone concentration also declined significantly with increasing body mass index (r = -0.519, P = 0.023). Although there were strong combined effects of age, body mass index, and LH secretory burst amplitude on serum free testosterone concentrations (P = 0.0006, multi-r value 0.820), LH secretory burst amplitude was the most prominent single determinant of blood androgen concentrations.


The results of various modalities of treatment of well differentiated thyroid carcinomas: a retrospective review of 1599 patients.

This study analyzed the impact of prognostic variables of age, sex, histopathological diagnosis, extent of disease at diagnosis, and surgical intervention on well differentiated thyroid carcinoma and how surgical treatment, radioactive iodine, and radiotherapy influence the patients' outcomes. There have been 1599 patients with well differentiated thyroid cancer treated and followed at the University of Texas M.D. Anderson Cancer Center from 1948 to 1989. The median follow-up for all patients was 11.0 yr, with the maximum follow-up being 43 yr and the minimum follow-up being 1 yr. The patients were predominantly female (2.3:1), with papillary (81%) and intrathyroidal carcinomas (42%) at the time of diagnosis. Sixty-six percent of the patients had a total thyroidectomy, 7% received external radiotherapy, and 46% had radioactive iodine as part of the treatment of the original disease; the overall recurrence rate was 23%, and the death rate was 11%. This study showed that treatment with radioactive iodine was the single most powerful prognostic indicator for increased disease-free interval (P less than 0.001) and that its use significantly increased survival as well. No benefit was obtained from treatment with external radiotherapy. Children had the best overall survival, but of the adult patients, females who had intrathyroidal papillary disease treated with total thyroidectomy, who had been given radioactive iodine, and whose disease had been diagnosed between 20-59 yr of age had the best prognosis.


3,3',5'-Triiodothyronine inhibits collagen-induced human platelet aggregation.

To clarify further the activity of rT3, we examined the effect of rT3 on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rT3, T4, T3, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rT3 inhibited by 50% collagen-induced platelet aggregation was 30 +/- 4 (mean +/- SE) mumol/L. rT3, T4, and T3 did not differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT3 inhibited collagen-induced phosphorylation of the 20-kilodalton protein (myosin light chain) in platelets. In contrast, rT3 did not inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)- or thrombin-induced platelet aggregation and inhibited only minimally TPA-induced 40-kilodalton protein phosphorylation. These results suggest that rT3 inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT3.


Effects of iodine on thyroid status of fetus versus mother in treatment of Graves' disease complicated by pregnancy.

To investigate the effect of maternal iodine therapy for Graves' disease on fetal thyroid, we examined serum free T4 (FT4) and TSH levels in the fetus vs. those in the mother. Patients who were severely thyrotoxic were not included. Cord and maternal sera were tested at delivery in 35 patients with Graves' disease treated with iodine alone during pregnancy (6-40 mg daily). At the initiation of therapy, the mothers were at 11-37 weeks gestation, and FT4 levels ranged from 28.3-65.8 pmol/L. At delivery, maternal FT4 values ranged from 9.3-42.0 pmol/L, slightly above normal in 22 of the 35 mothers and normal in the other 13. Fetal FT4 levels were above the normal range occurred significantly less often than maternal levels (2 in 35; P less than 0.001), and no fetus had FT4 below normal. In the 13 mothers with normal FT4 levels, all fetal FT4 levels were normal; the fetal TSH level was above normal in 1 and normal in the remainder. A significant correlation was found between cord and maternal FT4 levels (P less than 0.05). In 12 of the 35 mothers, FT4 levels rose after a transient fall during iodine administration. The correlation of cord FT4 and maternal FT4 was closer when these 12 cases were excluded (P less than 0.001). Neither the dose of iodine nor the duration of therapy correlated with thyroid function in fetuses or mothers. Fetal TSH binding inhibitory antibody values strongly correlated with maternal TSH binding inhibitory antibody values (P less than 0.001). These findings indicate that 1) in the treatment of hyperthyroidism due to Graves' disease, iodine seldom if ever exposes the fetus to the risk of hypothyroidism; 2) the fetal thyroid is influenced by the same stimulatory and inhibitory factors as the maternal thyroid; and 3) escape from the inhibitory effects of iodine occurs less often in fetuses than in mothers, which may account at least in part for the lower thyroid status in the fetus compared to that in the mother.


Effect of hyperthyroidism on the transit of a caloric solid-liquid meal through the stomach, the small intestine, and the colon in man.

Gastric emptying, mouth-to-cecum transit, and whole gut transit of a solid-liquid meal was measured in 20 hyperthyroid patients and in 30 control subjects by using scintigraphic techniques, the hydrogen breath test, and stool markers. In the hyperthyroid patients predefined gastrointestinal symptoms were determined and related to gastrointestinal transit. There was no significant overall difference of gastric emptying between the hyperthyroid patients and the control subjects, whereas both mouth-to-cecum transit time (mainly reflecting small intestinal transit) and whole gut transit time (mainly reflecting large intestinal transit) were significantly accelerated in hyperthyroid patients as compared to the control subjects (P less than 0.005). Mouth-to-cecum transit in hyperthyroid patients with diarrhea tended to be more rapid than in those without diarrhea (P = 0.094) and the T3 concentrations were inversely correlated with mouth-to-cecum transit time. It is concluded that in thyrotoxicosis 1) small and large intestinal transit is accelerated, while gastric emptying remains unchanged and 2) rapid intestinal transit is likely to be one factor among others implicated in the generation of diarrhea.


Vasopressin, corticotrophin-releasing factor, and pituitary adrenal responses to acute cold stress in normal humans.

Acute cold stress is a consistent stimulus to ACTH secretion in rats yet inhibits arginine vasopressin (AVP) in both rats and humans. We have studied the interrelationships of AVP, corticotrophin-releasing factor, and atrial natriuretic factor (ANF) in the hypothalamo-pituitary-adrenal response to acute cold stress in normal humans. Six healthy male volunteers deprived of food and fluid for 6 h, and minimally clothed, were studied in the early afternoon. After a 30-min period at 22 C, subjects were exposed to cold stress (4 C for 30 min), followed by a 30-min equilibration period at 22 C. By the end of the period of cold exposure there was a fall in plasma volume of 7.8 +/- 1.4% (mean +/- SEM), a significant increase in both systolic blood pressure (P = 0.0001) and in plasma norepinephrine level (P = 0.0001), but no change in plasma epinephrine or in plasma ANF. Plasma AVP levels fell significantly (P less than 0.01) to reach a nadir at 5-10 min after cold exposure before returning to baseline levels. A significant fall in plasma cortisol levels occurred during the first 15 min of the baseline period and remained stable thereafter. No significant changes in plasma corticotrophin-releasing factor or ACTH occurred. These results suggest that cold inhibition of AVP release, presumably via afferent baroreceptor pathways, may act to reduce the response of the corticotrophs to a potentially noxious stimulus. Inhibition of AVP and/or ACTH during acute cold exposure are not dependent upon an increase in plasma ANF.


Activin-A inhibits progesterone production by macaque luteal cells in culture.

Since inhibin is produced during the luteal phase of the menstrual cycle in women and nonhuman primates, the primate corpus luteum (CL) may be a local site of inhibin/activin action. This study was designed to determine whether inhibin or activin altered steroidogenesis by macaque luteal cells in vitro. Luteal cells were obtained by enzymatic dispersion of CL from rhesus monkeys at midluteal phase of the menstrual cycle. Cells (2 x 10(4)/0.2 mL) were cultured in wells coated with extracellular matrix from bovine corneal endothelial cells in Dulbecco's modified Eagle's medium/F-12 medium (1:1 vol/vol) + insulin (2 ng/mL), transferrin (5 ng/mL), H2SeO3 (0.25 nmol), and aprotinin (10 micrograms/mL). Various concentrations (0-400 ng/mL) of recombinant human-inhibin-A, recombinant human-activin-A or human CG (hCG) (100 ng/mL; CR123) alone or in combination with inhibin or activin were added to the culture media (n = 5 Exp). Media were changed daily for 4 days and progesterone (P) concentrations were determined by RIA. Inhibin exposure did not alter P levels compared to that of control (untreated) cultures. In contrast, activin (10-400 ng/mL) suppressed P production (P less than 0.05) below controls and inhibin-treated cultures by days 3 and 4. Exposure to hCG increased P levels throughout culture (9 x control levels by day 4; P less than 0.05). hCG-stimulated P production was unaltered by inhibin, whereas activin (50-400 ng/mL) reduced (maximal inhibition of 40%; P less than 0.05) hCG-stimulated P production by day 4 of culture. Cell number on day 4 was not altered by any dose of inhibin or activin, but the number of cells staining for 3 beta-hydroxysteroid dehydrogenase was reduced (P less than 0.05) by 32.9 +/- 2.6% in activin-treated cultures. Since P levels declined during culture in all treatment groups, in a second series of experiments (n = 4), luteal cells were cultured for 4 days with or without hCG (100 ng/mL) and low density lipoprotein (LDL; 100 micrograms/mL) +/- 0-400 ng activin/mL. P production in the presence of hCG+LDL was greatly enhanced compared to other treatment groups and was sustained during days 2-4 of culture. Activin at doses of 50-400 ng/mL suppressed (maximal inhibition of approximately 35%; P less than 0.05) hCG+LDL-stimulated P production on days 3 and 4. These results suggest that the primate CL is a target for activin action to suppress luteal cell activities, including gonadotropin-regulated, lipoprotein-mediated steroidogenesis.


Fat mass is an important determinant of whole body bone density in premenopausal women but not in men.

We recently reported that total body fat mass is the principal determinant of bone density in normal postmenopausal women. We have now reexamined the relationships among these variables and lean mass in 68 healthy premenopausal women and 51 men. Areal bone density (BMD), fat mass, and lean mass were measured in total body scans by dual-energy, x-ray absorptiometry. In women, BMD was correlated with weight (r = 0.69), fat mass (r = 0.60), and lean mass (r = 0.55). In men, the respective correlations were 0.56, 0.26 (NS), and 0.51. Multiple regression analysis confirmed a codependence of female BMD on fat and lean masses, whereas male BMD was related only to lean mass. Because BMD is an areal not volumetric density, it is dependent on body size. The analysis was therefore repeated using BMD/height as an index of "true" density. Correlations with fat mass were little changed but those with lean mass were reduced (women) or eliminated (men). By multiple regression, female BMD/height was related to fat mass alone, and in men there was a borderline effect of fat (P = 0.05) but none of lean mass. As a second method to exclude a scale artifact, fat mass was expressed as percent body weight. It was related to BMD (r = 0.48) only in women. It is concluded that bone density is closely related to fat mass in premenopausal women, but less so in men. In both sexes, apparent relationships between BMD and lean mass are artifacts attributable to the use of areal density (which is dependent on body size) as a surrogate for volumetric density. The mechanism of this fat-bone density relationship is an important question to be addressed in bone biology.


Rapid stimulation of human chorionic gonadotropin secretion by interleukin-1 beta from perifused first trimester trophoblast.

Placental trophoblast has been implicated as a major source of interleukin-1 beta (IL-1 beta), a cytokine that mediates immunological responses in the body. This study evaluated the effect of IL-1 beta on hCG secretion from 8- to 12-week-old placental trophoblast. Physically dissociated trophoblast cells collected from multiple placentae were cultured on carrier beads and loaded into chambers in a perifusion system. Medium was perifused through the chambers, and effluent was collected and assayed for hCG. Basal hCG secretion was not dependent on exogenous IL-1 beta or GnRH, but varied between mixed placental preparations and increased with duration of culture. Therefore, hCG secretion was expressed as a percentage of mean basal hCG secretion for any given chamber. IL-1 beta (10(-9) M) stimulated a rapid and transient hCG secretory response. hCG release increased by approximately 150% (P less than 0.05; n = 5) in response to the cytokine, but lower concentrations (10(-10) and 10(-11) M) were ineffective (P greater than 0.05; n = 3 each). GnRH stimulated hCG secretion by approximately 80% (P less than 0.05; n = 6). The hCG secretory profiles in response to IL-1 beta and GnRH were similar. Combined treatment with equimolar (10(-9) M) IL-1 beta and GnRH increased hCG secretion by approximately 150% (P less than 0.05; n = 5), stimulating hCG secretion as effectively as either hormone alone (P greater than 0.05). The stimulatory effect of GnRH on hCG secretion was blocked by the concomitant presence of a GnRH antagonist, Nal-Glu-GnRH (P less than 0.05; n = 5). However, simultaneous treatment with IL-1 beta and Nal-Glu-GnRH did not affect IL-1 beta-stimulated hCG secretion (100.5 +/- 3.6 vs. 162.9 +/- 10.2%; P less than 0.05; n = 7). The data suggest that IL-1 beta and GnRH stimulated a near-maximal physiological hCG secretory response, possibly through different receptor types. Alternatively, these two hormones may share a common signal transduction pathway, or IL-1 beta may influence a step distal to the coupling of GnRH to its receptor in the placental trophoblast.


Lipolysis and gluconeogenesis from glycerol are increased in patients with noninsulin-dependent diabetes mellitus.

The rate of lipolysis (glycerol Ra), gluconeogenesis from glycerol, and its contribution to overall hepatic glucose production (glucose Ra) were determined in 10 patients with noninsulin-dependent diabetes mellitus (NIDDM) [body mass index (BMI) 27.2 +/- 1.0 kg/m2, fasting plasma glucose 10.3 +/- 1.2 mmol/L], and in 6 matched control subjects (BMI 27.3 +/- 1.1 kg/m2, fasting plasma glucose 5.3 +/- 0.3 mmol/L) using infusions of [3-3H]glucose (0-600 min) and [U-14C]glycerol (360-600 min). Glycerol Ra was increased in the patients with NIDDM (120 +/- 16 mumol/m2.min) compared to the normal subjects (84 +/- 9 mumol/m2.min, P less than 0.05). Gluconeogenesis from glycerol was 1.7-fold higher in the patients (96 +/- 16 mumol/m2.min) than in the normal subjects (56 +/- 10 mumol/m2.min, P less than 0.05), and explained 9 +/- 1% and 7 +/- 1% (NS) of total glucose Ra in patients with NIDDM and normal subjects, respectively. To determine whether these abnormalities are more pronounced in overweight patients with NIDDM, glucose and glycerol Ra were also determined in 5 obese patients with NIDDM (BMI 36.4 +/- 1.0 kg/m2, fasting plasma glucose 11.3 +/- 1.3 mmol/L). Glycerol Ra (154 +/- 26 mumol/m2.min) was again higher than in the normal subjects (P less than 0.05) but not different from that in the less obese patients with NIDDM. The rate of gluconeogenesis from glycerol (159 +/- 20 mumol/m2.min) was significantly higher in the obese than in the less obese patients with NIDDM (P less than 0.05) but its contribution to total glucose Ra (10 +/- 1%) was similar to that in the less obese patients with NIDDM. When all data were analyzed together, gluconeogenesis from glycerol (r = 0.57, P less than 0.01) but not lipolysis (r = 0.02, NS) correlated with the percentage of lipolysis diverted toward gluconeogenesis suggesting that the rate of gluconeogenesis from glycerol is regulated by intrahepatic mechanisms rather than by glycerol availability. Neither the rate of lipolysis nor the rate of glycerol gluconeogenesis correlated with BMI, serum triglyceride, or insulin concentrations. We conclude that gluconeogenesis from glycerol is increased in patients with NIDDM. This increase appears to be the consequence of both accelerated lipolysis and increased intrahepatic conversion of glycerol to glucose.


Defect in insulin action on expression of the muscle/adipose tissue glucose transporter gene in skeletal muscle of type 1 diabetic patients.

Recently several members of the glucose transporter family have been identified by molecular cloning techniques. We determined the effect of a 4-h insulin infusion on the expression of the muscle/adipose tissue (GLUT-4) glucose transporter mRNA and protein in 14 insulin-treated type 1 diabetic patients and 15 matched nondiabetic subjects. GLUT-4 mRNA and protein concentrations were determined in muscle biopsies taken before and at the end of the insulin infusion during maintenance of normoglycemia. In response to insulin, muscle GLUT-4 mRNA increased in the nondiabetic subjects from 24 +/- 3 to 36 +/- 4 pg/microgram RNA (P less than 0.001) but remained unchanged in the insulin-resistant diabetic patients (24 +/- 2 vs. 26 +/- 2 pg/microgram RNA, before vs. after insulin). The glucose transporter protein concentrations were similar in the basal state and decreased by 21 +/- 7% (P less than 0.02) in the normal subjects but remained unchanged in the diabetic patients. The increase of the GLUT-4 mRNA and the decrease in the GLUT-4 protein correlated with the rate of glucose uptake [correlation coefficient (r) = -0.55, P less than 0.01, and r = -0.44, P less than 0.05, respectively]. We conclude that the insulin response of both the GLUT-4 glucose transporter mRNA and protein are absent in skeletal muscle of insulin-resistant type 1 diabetic patients. Thus, impaired insulin regulation of glucose transporter gene expression can be one of the underlying mechanisms of insulin resistance in type 1 diabetes.


Maternal and neonatal thyroid function at birth in an area of marginally low iodine intake.

Thyroid function was evaluated in cord serum of healthy full-term newborns and compared to that of mothers immediately after parturition. The study was carried out in an area without overt iodine deficiency, but with a marginal iodine supply (less than 100 micrograms/day in 80% of women). The aim of the study was to delineate the interrelationships between the thyroid statuses of mother and child at birth. Maternal thyroid function was characterized at delivery by relative hypothyroxinemia; increased T3/T4 ratios, indicating preferential T3 secretion; slightly increased TSH levels within the normal range in 97% of women; increased serum thyroglobulin (TG) values, which were above normal in 60% of women; and also goiter formation in almost 10% of women. The findings indicated glandular stimulation and confirmed our earlier reports that pregnancy constitutes a stress for the maternal thyroid economy, enhanced by the limited availability of iodine in the diet. By contrast, newborns showed a strikingly distinct pattern: there was no relative hypothyroxinemia and free T4 levels were significantly higher than in the respective mothers (19.4 vs. 14.7 pmol/L; P less than 0.001). In spite of these differences, however, mean neonatal TSH and TG levels were significantly higher than maternal values, respectively 6.0 vs. 1.9 mU/L for TSH (P less than 0.001) and 70 vs. 40 micrograms/L for TG (P less than 0.001). Furthermore, neonatal TG and TSH levels increased in parallel and were highly correlated with maternal data, suggesting a regulatory link between both thyroid economies. The results suggested that the common regulatory link is the limited availability of the iodine supply. In conclusion, the present study demonstrates that even in conditions with a marginally low iodine intake, pregnancy constitutes a stimulus for both the maternal and newborn thyroids. Changes in both groups are associated and the abnormalities in TSH and TG are amplified in the newborns. The TSH and TG alterations at birth in full-term healthy newborns, associated with similar alterations in maternal thyroid function, provide evidence for a common stimulatory factor, relative iodine deficiency. The data emphasize the hypersensitivity of neonatal thyroid function to marginal iodine deficiency and point to the need to increase the iodine supply in groups at risk, such as women during pregnancy, and also newborns in the perinatal period.


Regulation of pulsatile growth hormone secretion by fasting in normal subjects and patients with acromegaly.

In acromegaly, GH hypersecretion occurs despite elevated insulin-like growth factor-I (IGF-I) levels, implying defective IGF-I feedback. To study the possible mechanisms of defective IGF-I negative feedback in acromegaly, we assessed parameters of pulsatile GH secretion during fasting-induced decrease in plasma IGF-I. Seven patients with active acromegaly and six normal controls were fasted for 6 days and GH secretory profiles were obtained by frequent (every 10 min) blood sampling for 24 h and analyzed by Cluster. Fasting resulted in similar decreases in IGF-I, body weight, and blood glucose levels, and increases in free fatty acid and beta-hydroxybutyrate in all subjects. Normal subjects showed increases in 24-h total and pulsatile GH production, GH pulse frequency, maximal pulse amplitude, interpulse and nadir levels, implying suppression of hypothalamic somatostatin secretion and increase in GH-releasing hormone (GHRH) pulse frequency. In acromegalic patients, GH (and, by inference, GHRH) pulse frequency was unchanged. Three patients had increases in GH production, interpulse, and nadir levels similar to the normals while the other four had no change or paradoxical decreases in these parameters. Percentage change in GH production was highly correlated with percentage change in interpulse and nadir levels in both normals and patients. Mean GH response to GHRH (0.33 micrograms/kg iv) did not change significantly in any group as a result of fasting. Our data suggest that in healthy humans IGF-I negative feedback on GH secretion involves suppression of GHRH pulse frequency. GH (and, by inference, GHRH) pulse frequency is resistant to decrease in IGF-I in acromegaly, suggesting that lowered sensitivity of GHRH neurons to IGF-I may be the mechanism of high GH pulse frequency in this disease.


Differing responses of plasma bioactive and immunoreactive follicle-stimulating hormone and luteinizing hormone to gonadotropin-releasing hormone antagonist and agonist treatments in postmenopausal women.

Plasma bioactive (B) and immunoreactive (I) FSH and LH were measured every 10 min for 8 h in the same postmenopausal women in a three-phase study: 1) during normal pulsatile gonadotropin secretion (basal study; n = 8), 2) 8 h after a single injection of a GnRH antagonist (5 mg Nal-Glu, sc; n = 5), and 3) 21 days after a GnRH agonist injection (D-Trp6, 3.75 mg depot preparation, im; n = 7). I-FSH and I-LH were measured by monoclonal antibody immunoradiometric assays. B-FSH and B-LH were measured in selected samples with the immature rat granulosa cell and mouse interstitial cell assays, respectively. Significant pulsatility of B- and I-FSH and LH was demonstrated in the basal samples, but only the B/I ratio of LH was slightly elevated within the secretion peaks. After GnRH antagonist treatment, I-FSH decreased from a mean pretreatment level of 55.7 +/- 7.8 IU/L by 26% (P less than 0.001), and B-FSH from 313.8 +/- 61.9 IU/L by 44% (P less than 0.01). The B/I ratio decreased from 6.4 +/- 1.7 to 4.5 +/- 1.0 (P less than 0.05). After agonist treatment, the I- and B-FSH levels decreased by 92% and 83% (P less than 0.0001), respectively, but the B/I ratio increased to 17.3 +/- 4.7 (P less than 0.05). The concentrations of I- and B-LH decreased by 75% and 80%, respectively (P less than 0.001), after antagonist treatment. After agonist treatment, I-LH decreased by 92%, and B-LH by 93% (P less than 0.0001). No changes in the B/I ratios of LH were found after either treatment. In conclusion, no changes were found in the quality of circulating LH during the treatments, whereas the antagonist treatment decreased and the agonist treatment increased the B/I ratio of FSH. These findings provide further evidence that the qualitative responses of FSH and LH to treatment with the same GnRH analog are different, and that the suppressive mechanisms of GnRH antagonist and agonist action on gonadotropin secretion are different.


Preclinical Cushing's syndrome in adrenal "incidentalomas": comparison with adrenal Cushing's syndrome.

Adrenal tumors are usually diagnosed by clinical symptoms of hormone excess. The increasing use of ultrasound and computed tomography results in the detection of a substantial number of incidentally discovered adrenal tumors. Most of these tumors are nonfunctional adrenocortical adenomas, but a few cases of subclinical cortisol production in "incidentalomas" have been reported. We investigated prospectively the prevalence of autonomous cortisol production in 68 patients (44 females and 24 males, aged 25-90 yr) with adrenal incidentalomas at our institution. As a screening procedure all patients with incidentalomas underwent an overnight dexamethasone suppression test (1 mg). Patients who failed to suppress serum cortisol below 140 nmol/L (5 micrograms/dL) underwent more comprehensive studies (prolonged dexamethasone suppression test, determination of the diurnal rhythm of cortisol secretion in saliva, and CRH stimulation test). Eight patients (12% of all patients with incidentalomas; 5 females and 3 males, aged 25-71 yr) were finally identified as having cortisol-producing tumors, and the findings in these patients were compared with those of overt Cushing's syndrome in 8 patients (8 females, aged 26-50 yr) suffering from cortisol-producing adrenal adenomas. The tumor size of patients with cortisol-producing incidentalomas ranged from 2-5 cm. No specific signs and symptoms of hypercortisolism were present, but arterial hypertension (seven of eight subjects), diffuse obesity (four of eight subjects), and noninsulin-dependent diabetes mellitus (NIDDM; two of eight subjects) were frequently observed. Baseline cortisol levels were in the normal to upper normal range, whereas baseline ACTH levels were suppressed in five of the eight patients. In none of the patients was serum cortisol suppressible by low dose or high dose dexamethasone. The ACTH and cortisol responses to CRH were normal in two, blunted in one, and suppressed in four patients. Unilateral adrenalectomy was performed in seven patients and resulted in temporary adrenal insufficiency in four of them. After surgery, improvement of arterial hypertension, a permanent weight loss in obese subjects, and a better metabolic control of NIDDM were noted in the majority of patients. The following conclusions were reached. Incidentally diagnosed adrenal tumors with pathological cortisol secretion in otherwise clinically asymptomatic patients are more frequently observed than previously assumed. Adrenocortical insufficiency is a major risk in these patients after adrenalectomy. After surgery, hypertension, obesity, and NIDDM may improve. Patients with asymptomatic adrenal incidentalomas, therefore, should be screened for cortisol production by means of an overnight dexamethasone suppression test.


Body composition in adult growth hormone-deficient men, assessed by anthropometry and bioimpedance analysis.

Anthropometry and bioimpedance analysis (BIA) were used to assess body composition in 42 GH-deficient (GHD) adult males (mean age, 27.2 +/- 4.7 yr). During childhood, all patients had received GH treatment for a mean period of 8.4 +/- 3.8 yr. At the start of this study, GH therapy had been discontinued for a mean period of 7.5 +/- 4.5 yr. Eighteen patients had isolated GH deficiency (I-GHD). Twenty-four patients had multiple pituitary hormone deficiencies (M-PHD), substituted adequately. Compared to age- and sex-matched controls, the sum of skinfolds measured at 7 different sites was significantly higher in I-GHD and M-PHD patients [controls, 73.1 +/- 25.4 mm; I-GHD patients, 102.1 +/- 37.7 mm (P less than 0.001); M-PHD patients, 126.8 +/- 35.4 mm (P less than 0.001)]. The increase in sc fat deposition was most pronounced on the trunk, particularly in the breast and abdominal area. Total body muscle mass was significantly lower in GHD patients (P less than 0.001). In patients, body muscle mass and plasma somatomedin-C level were positively correlated (r = 0.43; P less than 0.005). Total body resistance measured by whole body BIA was significantly higher in the patient group and was negatively correlated with plasma somatomedin-C (r = -0.53; P less than 0.001). The high resistance values observed in GHD patients could only in part be explained by their lower lean body mass. The most important cause, however, was an increase in specific electrical resistance of the lean body mass (LBM), reflecting relative dehydration. We conclude that GH deficiency in adult males is associated with an increase in sc fat and a decrease in body muscle mass. In addition, there is a qualitative change in LBM. The BIA data indicate that in these patients, the hydration state of the LBM is lower than normal, due to a decrease in extracellular water.


In vitro stimulation of placental progesterone production by 19-nortestosterone and C19 steroids in early human pregnancy.

To explore the regulatory mechanism at the critical period of the luteal-placental shift, the effects of various steroids and peptides on the production of progesterone by placental explants at 7-10 weeks were studied. Androstenedione increased progesterone production 3-fold at a concentration of 1 mumol and more than 20-fold at 18 mumol. 19-Nortestosterone (1-18 mumol) stimulated progesterone production 10- to 100-fold. 5 alpha-Androstane-3 beta,17 beta diol (1-18 mumol) stimulated progesterone production about 2- to 5-fold while its 3 alpha isomer (1-6 mumol) increased it 2-fold. Estrone, estradiol, and estriol up to a concentration of 30 mumol had no effect. Dehydroepiandrosterone sulfate (to 36 mumol), androst-5-ene-3 beta,17 beta diol (1-6 mumol), 5 beta-androstane-3 alpha,17 beta diol (1-6 mumol), and dihydrotestosterone (1-12 mumol) had no effect. Cortisol and dexamethasone (up to 12 mumol), hCG (20,000 IU/L), GnRH (4 mumol), and ACTH 1-24 (20 mumol) also had no effect. Thus, of all the compounds tested, only 19-nortestosterone and, to a lesser extent, androstenedione, 5 alpha-androstane-3 beta,17 beta diol, and 5 alpha-androstane-3 alpha,17 beta diol stimulated progesterone production in early pregnancy; at term, only 5 alpha-androstane-3 beta,17 beta diol was stimulatory. 19-Nortestosterone was found to be less efficiently aromatized compared to other androgens; since it is also known to be present in blood from pregnant women and thought to be made in the placenta, the stimulation observed may be a paracrine effect. These observations suggest that C18 and C19 steroids may be important in the regulation of progesterone synthesis by the human placenta in early pregnancy.


Genetic linkage analysis in familial benign hypercalcemia using a candidate gene strategy. I. Studies in four families.

Despite extensive study since the first report of familial benign hypercalcemia (FBH, or hypocalciuric hypercalcemia) in 1972, there is no evidence of the specific abnormal gene product. FBH is highly suitable for either a candidate gene or a reverse genetics approach to localizing the genetic abnormality, because it is inherited in an autosomal dominant pattern, is highly penetrant, does not affect survival, and can be diagnosed in families with readily available measurements. Importantly, several candidate genes have been cloned and mapped. Therefore, we collected blood samples and extracted leukocyte DNA from 94 members of 4 families with well documented FBH (44 affected, 45 unaffected, and 5 unclassifiable). We digested the DNA samples with various restriction endonucleases, conducted standard Southern blotting, and searched for restriction fragment length polymorphisms for the following candidate genes (probe names in parentheses): multiple endocrine neoplasia (MEN) type 1 (pMCMP.1, pHBI59, p3C7, and pTHH26), MEN 2a (MCK2 and cTB14.34), basic fibroblast growth factor (pHFL1-7), (Ca2+,Mg2+)ATPase isoform 4 (hPMCA4), membrane Na/Ca exchanger (cNC28 M-A), PTH (pPTH-LF), and calbindin-D28K (pSKCalb). In addition, we used the anonymous variable number tandem repeat marker pYNH24 to verify pedigree structures by excluding misinheritances. Data were analyzed using the Linkage program. For none of the genes was there significant linkage with the FBH trait; logarithm of odds scores ranged from -1.3 to -26.0 at a recombination fraction of 0.001, and from 0.6 to -5.6 at a recombination fraction of 0.10. We conclude that FBH is unrelated to the MEN syndromes and is not caused by mutations in any of the calcium-regulating or -binding proteins or growth factors studied thus far.


Growth hormone-binding proteins of human serum: developmental patterns in normal man.

The binding of GH by a low affinity binding protein (LA-BP) was measured from birth into adulthood and compared with binding of a high affinity binding protein (HA-BP) in human serum. Pooled serum samples for each year of age from birth to 16.5 yr were formed from 2500 separate samples and assayed for both binding proteins using a Sephadex chromatographic method. Individual samples in this age range and those from adults were assayed in a similar manner. Binding of [125I]GH was minimal by both binding proteins in cord blood (binding by LA-BP, 2.77 +/- 0.32%; binding by HA-BP, 2.58 +/- 0.35% mean +/- SEM). A 4-fold increase to maximal binding for LA-BP occurred by the age of 5 yr and remained relatively constant through adolescence, except for a transient decrease at puberty. From 16.5-20 yr of age, binding by LA-BP decreased to a level no greater than that seen at birth. Binding by the HA-BP, which increased 6- to 8-fold reached maximal binding between 23-25 yr of age. Binding by HA-BP did not decrease in adults between the ages of 20-30 yr. Whereas pregnancy increased LA-BP activity, GH binding by HA-BP was unaltered.


A circadian rhythm of serum follicle-stimulating hormone in women.

While a nocturnal decline in serum LH levels in the early follicular phase of the menstrual cycle has been well established, a diurnal variation in serum FSH levels in women has not been demonstrated. We addressed this issue by determining serum LH and FSH levels at 15-min intervals for 24 h in the early follicular phase (EFP; n = 16) and late follicular phase (LFP; n = 10) of the menstrual cycle and in postmenopausal women (PMW; n = 10). Serum estradiol was simultaneously measured at hourly intervals. As expected, EFP, but not LFP and PMW, women had a 15% nocturnal decline (P less than 0.01) in transverse mean LH levels compared to values in the daytime hours. In contrast, nocturnal FSH transverse mean values were significantly lower than daytime values in all groups studied, demonstrating an 18% decline in EFP (P less than 0.001), a 17% decline in LFP (P less than 0.00001), and a 4.3% decline in PMW (P less than 0.01). Cosinor analysis revealed a circadian rhythm for FSH, with acrophases in the afternoon and nadirs at night in all three groups. The circadian amplitudes were 1.43 +/- 0.22, 1.02 +/- 0.16, and 8.42 +/- 1.31 IU/L for EFP, LFP, and PMW, respectively. The EFP nocturnal decline in LH did not conform to a cosine rhythm. A diurnal variation in estradiol was not present in any of the groups of women. These data constitute the first demonstration of a robust circadian rhythm of serum FSH in women. The comparable timing of the acrophase in all groups of subjects and its presence in the postmenopausal years suggest a central, rather than peripheral, feedback mechanism(s) for the circadian rhythmicity. This observation provides strong evidence for a dissociation in the hypothalamic regulation of pituitary LH and FSH secretion in women. The circadian peak and nadir of circulating FSH may prove to be determining for appropriate follicular development.


Combined effects of RU486 and tamoxifen on the growth and cell cycle phases of the MCF-7 cell line.

The antiproliferative effect of RU486 and its effect combined with tamoxifen on the growth and cell cycle kinetics parameters on the MCF-7 human carcinoma cells were investigated. When MCF-7 cells in the exponential growth phase were treated with RU486 (10(2) nmol/L), a time-dependent cell growth inhibition was observed which was significant 5 days after the beginning of treatment. This inhibition was accompanied by a time- and dose-dependent decrease in the percentage of S and G2-M phase cells and a concomitant increase in the percentage of cells in the G0/G1 phase of the cell cycle. With tamoxifen (10(5) pmol/L), growth inhibition was obtained after 4 days of treatment of cells, and the blockage of the cell cycle occurred in the G0/G1 phase. In the case of simultaneous treatment of MCF-7 cultures with RU486 (10(2) nmol/L) and tamoxifen (10(5) pmol/L), we observed a synergistic inhibitory effect on the proliferative rate for short treatment (less than or equal to 3 days), whereas RU486 or tamoxifen alone had no effect. For the intermediate treatment (4 days), the combined effect of RU486 and tamoxifen was not significant compared to the effect of tamoxifen alone. For the long treatment (greater than 4 days), there were no differences between the number of cells in the treated cultures under different experimental conditions, but all were inhibited compared to those in control cell cultures. This simultaneous treatment of cells does not induce any change in the distribution of cells in the different cell cycle phases compared to tamoxifen percentages. These results suggest that RU486 is a cell cycle phase-specific growth inhibitory agent, and a combination of antiestrogen/antiprogestin should be considered as a possible improvement in breast cancer endocrine therapy.


A prospective trial of phosphate and 1,25-dihydroxyvitamin D3 therapy in symptomatic adults with X-linked hypophosphatemic rickets.

Current treatment of X-linked hypophosphatemia (XLH) employs the combined administration of oral phosphate and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Although this drug regimen significantly improves the clinical course of the disease in children, the value of medical treatment in symptomatic adults with XLH has not been established. We, therefore, investigated the clinical, biochemical, and histological responses to phosphate and 1,25-(OH)2D3 in 16 symptomatic adult patients with XLH followed for a mean of 4.2 yr. Eighty-seven percent of the patients had an improvement in bone or joint pain with therapy. There was a significant increase in mean serum phosphate (from 0.61 +/- 0.03 to 0.77 +/- 0.03 mmol/L) and urinary calcium excretion (from 2.45 +/- 0.38 to 4.39 +/- 0.44 mmol/day) with treatment. Pretreatment bone biopsies demonstrated findings characteristic of osteomalacia, including abnormally increased osteoid volume and decreased mineral apposition rates. Treatment was accompanied by a significant decrease in osteoid thickness as well as a reduction in mean osteoid volume. However, therapy did not completely normalize these parameters. Disease severity, as assessed by histomorphometric parameters, did not correlate with any pretreatment serum or urinary biochemical measurement, but did seem to correlate with symptom score. Although most patients tolerated therapy without difficulty, 1 patient developed tertiary hyperparathyroidism during treatment and renal insufficiency that progressed despite cessation of therapy. This study provides evidence that therapy with oral phosphate and 1,25-(OH)2D3 in symptomatic adults with XLH can result in significant clinical and histomorphometric improvement.


Does Graves' disease or thyrotoxicosis affect the prognosis of thyroid cancer.

Twenty-one patients who underwent surgical treatment for thyrotoxicosis and who were found at operation to have thyroid cancer are presented. Sixteen had Graves' disease and 5 had toxic nodular goiter. The group with Graves' is compared with 110 euthyroid patients with thyroid cancer who underwent their initial surgery in the same time period and who were of the same age (+/- 1 yr) and sex as the patients with Graves' disease. None of the thyrotoxic patients died during follow-up of 2-24 yr or developed subsequent metastases. The 1 patient with a local lymph node metastasis has not shown evidence of recurrence. Hypoparathyroidism appeared as a complication in only 1 patient. The size of tumors in the patients with Graves' disease was significantly smaller than in the euthyroid group. The course of the disease in both the patients with Graves' disease and the thyrotoxic group as a whole was relatively benign. This series does not support the recent suggestions that thyroid cancer in patients with Graves' disease is more aggressive than in either patients with toxic nodular goiter or euthyroid subjects. Patients with Graves' disease and thyroid cancer should be treated identically to other patients with thyroid cancer. Therapy should consist of total thyroidectomy followed by a postoperative 131I scan. Residual tissue or metastases found on the scan should be ablated with 6 GBq 131I. The patient should receive a suppressive dose of T4.


Ovulation and menstrual function of adolescent girls with central precocious puberty after therapy with gonadotropin-releasing hormone agonists.

Chronic GmRH agonist (GnRHa) administration has been shown to suppress pituitary-gonadal function in children with central precocious puberty (CPP), but long term data after the reactivation of gonadarche posttherapy are not yet available. This study evaluated the menstrual function of 46 girls with CPP who had been treated for at least 2 yr with GnRHa (deslorelin or histrelin, sc, daily) and were up to 7 yr posttreatment, including 21 postmenarcheal girls who collected weekly overnight urine samples for 12 consecutive weeks to assess rates of ovulation by urinary pregnanediol-3 beta-glucuronide measurements. Menarche occurred at age 12.1 +/- 1.0 yr (mean +/- SD), on the average 1.2 +/- 0.8 yr posttherapy (range, 0.1-4.3 yr). Menstrual cycle lengths became increasingly regular, with cycles of 25- to 35-day duration reported by 41% of the girls in the first year postmenarche and 65% of the girls studied 3 or more years postmenarche. Ovulation was demonstrated in 50% of the girls studied within 1 yr of menarche and in 90% of the girls studied 2 yr or more postmenarche, including 5 girls who reported pregnancies. The development of regular ovulatory menstrual function in these girls with CPP is in accord with previously documented patterns in normal adolescents. While these data provide further evidence supporting the safety of long term GnRHa therapy, continued studies will be necessary to characterize fully the reproductive function in CPP patients through adolescence and adulthood.


Organ-specific effects of tiratricol: a thyroid hormone analog with hepatic, not pituitary, superagonist effects.

Tiratricol has been used to suppress pituitary TSH secretion, with reported attenuation of extrapituitary thyromimetic effects. A randomized, double-blind trial was performed to define precisely the tissue-specific thyromimetic actions of tiratricol. Ten athyreotic patients, treated for thyroid carcinoma, were randomly assigned to receive L-T4 sodium 0.7 micrograms/kg daily and either tiratricol 10 micrograms/kg or placebo twice daily. The daily dose of L-T4 was increased by 25-50 micrograms increments until the TRH-stimulated TSH level was less than 0.1 mU/L. After measurement of biochemical and physiological parameters of thyroid hormone actions, patients crossed treatment groups. Patients required 46% less L-T4 to achieve equivalent TSH suppression when taking tiratricol. Hepatic effects were enhanced by tiratricol administration, with significant increases in sex hormone binding globulin and ferritin concentrations, 14% and 37%, respectively. Levels of serum cholesterol, LDL cholesterol, and apolipoprotein B were reduced by 7%, 10%, and 13%, respectively, during tiratricol therapy. Triglyceride levels also declined, but there were no changes of high density lipoprotein cholesterol or apolipoproteins AI and AII. Resting metabolic rate, body weight, urea nitrogen excretion, and symptoms did not differ between the two treatment regimens. Cardiovascular function, as reflected by mean arterial pressure and pulse wave arrival time, was not different during tiratricol therapy. Skeletal metabolic activity was affected by tiratricol, with marked elevation of osteocalcin without significant change in serum calcium, PTH, and urinary calcium and hydroxyproline excretion. Tiratricol has increased hepatic and skeletal actions of potential therapeutic value, but does not have enhanced thyromimetic activity specific to the pituitary gland.


Highest concentrations of prorenin and human chorionic gonadotropin in gestational sacs during early human pregnancy.

Prorenin is not only the biosynthetic precursor of renin; under certain circumstances in vitro prorenin exhibits reversible intrinsic renin activity and can form angiotensin from renin substrate with or without cleavage of the prosequence. Prorenin is the predominant form of renin synthesized by reproductive organs (ovary, chorion laeve of the placenta, uterine decidua). Its plasma concentrations increases 10-fold throughout pregnancy to 10-100 times that of renin; amniotic fluid prorenin concentration is even higher. No data are available of gestational fluid prorenin concentrations during early pregnancy. For the first 10 weeks there are two gestational cavities; the chorionic cavity then disappears and the smaller amniotic cavity becomes predominant. In this study we measured prorenin, renin, renin substrate and hCG in fluid aspirated from gestational sacs during the first trimester of gestation (predominantly chorionic) and during the second and third trimesters (amniotic). Seventeen patients had amniocentesis during the second or third trimester. Nine patients underwent selective abortion of multiple pregnancy at 7-12 weeks gestation. One patient underwent surgery at 5 5/7 weeks (26 days after conception) for a tubal pregnancy. Second and third trimester amniotic fluid prorenin maximum velocity (Vmax) (16 and 3 sacs, respectively) averaged 6,100 +/- 1,700 (SD) and 1,930 +/- 760 ng/mL.h, respectively (i.e. 1,700 and 540 ng/L.s). In gestational fluid collected before 8 weeks, prorenin Vmax was 10-fold higher, averaging 62,500 +/- 40,000 ng/mL.h (17,000 ng/L.s). The concentration was 140,000 ng/mL.h (39,000 ng/L.s) in the 5 5/7 week tubal pregnancy. In sharp contrast, at 10-12 weeks gestation (n = 3) prorenin Vmax was only 260 +/- 114 ng/mL.h (72 ng/L.s); human CG was also highest before 8 weeks (276,500 +/- 110,900 IU/L) and lowest at 10-12 weeks (1210 +/- 540 IU/L) with intermediate levels occurring later in pregnancy. This study shows that the highest biological levels of prorenin yet detected (close to 1 micrograms protein/mL) occur in gestational sacs in early pregnancy, consistent with a role for the renin-angiotensin system in embryonic development or placentation.


The inhibitory effects of müllerian-inhibiting substance on epidermal growth factor induced proliferation and progesterone production of human granulosa-luteal cells.

This study was undertaken to determine if Müllerian-inhibiting substance (MIS) could block basal and/or epidermal growth factor (EGF)-induced proliferation and progesterone production by cultured human granulosa-luteal cells. Cells from follicles of individual patients were pooled, counted, and aliquoted into Ham's F-10 medium containing 10% MIS-free female fetal calf serum at 37 C in 95% air and 5% CO2. After assessing viability, cells were counted on days 4, 8, 12, and 16 of culture. EGF was added every other day at 0.2, 2, and 20 ng/mL beginning on culture day 4. The greatest stimulatory effect of EGF on cell proliferation was observed at 20 ng/mL on days 12 and 16. EGF increased progesterone production per cell after 4 days exposure, but this effect was lost after 8 days. Granulosa-luteal cells were cultured with 0.2, 2, and 20 ng/mL immunoaffinity purified recombinant human MIS (rhMIS) or conditioned medium from Chinese hamster ovary cells transfected with the human MIS gene, beginning on culture day 4. rhMIS demonstrated its greatest inhibitory effect on cell proliferation at 20 ng/mL on day 16. The rhMIS decreased progesterone production per cell after 4 days exposure, but only in the higher doses. Maintaining EGF at 20 ng/mL and varying rhMIS yielded significant reduction in EGF-mediated proliferation and progesterone production per cell at 2 and 20 ng/mL rhMIS. These experiments demonstrate rhMIS inhibits basal and EGF-stimulated human granulosa-luteal cell proliferation and progesterone production.


Glycoprotein hormone alpha-subunit production in somatotroph adenomas with and without Gs alpha mutations.

Activating mutations of the Gs alpha subunit have been identified in a subset of somatotroph adenomas. The mutant form of the Gs alpha subunit causes persistent activation of adenylyl cyclase and consequently results in high intracellular levels of cAMP. Because cAMP is known to stimulate the synthesis of the glycoprotein hormone (GPH) alpha-subunit as well as GH, we examined somatotroph tumors with and without Gs alpha mutations for GPH alpha-subunit production. GPH alpha-subunit production was assessed in vivo by measuring serum hormone levels and in vitro by analyzing hormone secretion by cultured pituitary tumor cells. DNA was extracted from the pituitary tumors of 26 acromegalic patients. The Gs alpha gene was amplified by the polymerase chain reaction and screened for mutations at codons 201 and 227 using oligonucleotide specific hybridization. Nine of the 26 tumors (35%) had point mutations at Arg 201. Seven of these tumors contained a CGT to TGT mutation (Arg to Cys) and 2 contained a CGT to CAT mutation (Arg to His). No mutations were detected at codon 227. There were no significant differences in age, sex distribution, tumor size, or serum levels of GH or insulin-like growth factor-1 between the groups of patients with or was Gs alpha mutations. The mean serum level of the free GPH alpha-subunit was 1.9-fold higher in the group with Gs alpha mutations (0.48 +/- 0.37 micrograms/L) than in patients without mutations (0.25 +/- 0.17) (P less than 0.05). In pituitary tumor cell culture, 75% of somatotroph tumors with Gs alpha mutations secreted free GPH alpha-subunit into the media compared with 45% of tumors without Gs alpha mutations. The amount of GPH alpha-subunit secretion was 12-fold greater in the group of tumors containing the Gs alpha mutation (P less than 0.05). Immunocytochemical detection of the free GPH alpha-subunit was similar in the two groups of patients with 75% positive for the GPH alpha-subunit in tumors with Gs alpha mutations and 67% positive in tumors without mutations (P = 0.69). We conclude that GPH alpha-subunit production occurs in somatotroph tumors with and without Gs alpha mutations. The increased levels of GPH alpha-subunit secretion in vivo and in vitro suggest that the Gs alpha mutation may increase the amount of preexisting GPH alpha-subunit biosynthesis in the tumors, perhaps via activation of the cAMP pathway.


Plasma neurotransmitter profile during different phases of the ovulatory cycle.

The influence of the different phases of the menstrual cycle on platelet-poor plasma norepinephrine (NE) and serotonin (5HT) was examined in 17 normal volunteers. The examinations were performed consecutively during 3 phases of the ovulatory cycle: 1) follicular phase, 2) ovulation, and 3) luteal phase. This investigation was initiated after a preliminary study in 51 volunteers showed wide and consistent variations of plasma NE and 5HT during the different phases of the cycle. Since in this first group the determinations had not been performed consecutively in the same subjects, and the changes observed in the different phases of the cycle could reflect interpersonal variations, the determinations were performed consecutively in a second group, concomitantly with serum estradiol (E2) and LH measurements. The results showed a decrease in plasma 5HT from the follicular phase [144.3 +/- 69.3 nmol/L (+/- SD)] to ovulation (55.7 +/- 41.4; P less than 0.001) and a subsequent increase in the luteal phase (141.3 +/- 96.4; P less than 0.01). The nadir in plasma 5HT showed an inverse correlation with serum LH (r = -0.07). Plasma NE increased from the follicular phase (1226.5 +/- 475.1 pmol/L) to ovulation (1694.0 +/- 564.4; P = 0.027) and reached a maximum in the luteal phase (2335.0 +/- 728.2; P = 0.0034). This rise correlated positively with serum E2. In conclusion, plasma 5HT and NE vary with the different phases of the menstrual cycle. Plasma NE rises during ovulation and seems to to correlate positively with serum E2 levels. Plasma 5HT reaches a nadir during ovulation and correlates inversely with serum LH.


Alterations in spontaneous growth hormone (GH) secretion and the response to GH-releasing hormone in children with nonorganic nutritional dwarfing.

The effects of suboptimal nutrition on the spontaneous overnight GH secretion (SGHS) and the GH response to GHRH were studied. Sixteen patients with nonorganic nutritional dwarfing (ND) were compared with 25 healthy short children with familial short stature with or without constitutional growth delay (FC). The effects of puberty were also assessed. All patients underwent an overnight study to assess SGHS with serum GH levels sampled every 20 min for 12 h, and a GHRH stimulation test was administered. Pubertal ND children had a blunted SGHS with a mean overnight GH level of 4.9 +/- 1.1 micrograms/L, significantly less than the level of 6.2 +/- 1.8 micrograms/L of the pubertal FC children (P less than 0.05). Also, prepubertal ND patients had an area under the curve in GH secretion after GHRH which was greater than that of the pubertal ND patients (2483 +/- 1581 vs. 1600 +/- 1056, P less than 0.05). The peak GH response to GHRH in the prepubertal ND patients was also higher than that of the pubertal ND patients (51.8 +/- 22.1 micrograms/L vs. 22.5 +/- 15.4 micrograms/L, P less than 0.05). This study shows that the SGHS is attenuated in ND patients during puberty but their GH response to GHRH is increased before adolescence. These abnormalities may represent compensatory mechanisms to energy restriction and may increase our understanding of the poor growth seen in ND patients.


Long term follow-up of Cushing's disease treated with reserpine and pituitary irradiation.

Twenty adult patients with Cushing's disease treated with long term reserpine administration in combination with a single course of external pituitary irradiation were followed. Eleven patients experienced long term remissions of 15.5 +/- 8.9 (mean +/- SD) yr (55%) after a mean irradiation dose of 53.9 +/- 11.4 Gy and a mean duration of reserpine administration of 24.3 +/- 9.3 months. The long term remission rates of the patients irradiated with 50 Gy or less (n = 9; 45.0 +/- 7.1 Gy) and those with more than 50 Gy (n = 10; 61.3 +/- 3.3 Gy; excluding 1 irradiated with 66 Gy who developed brain necrosis) were 56% (5 of 9) and 60% (6 of 10), respectively, and did not differ significantly. There were no significant differences between the 2 groups with regard to the duration of reserpine administration or pretreatment clinical features. At the latest examination, regardless of the irradiation dose, all 9 patients with long term remission showed a higher level of plasma cortisol or 11-hydroxycorticosteroids in the morning than in the evening, normal suppressibility of plasma cortisol with overnight 1 mg dexamethasone (9 of 10), and well preserved responses of other pituitary hormones to various loading tests: normal responses of plasma ACTH to CRH (6 of 9), TSH (7 of 8), and PRL (5 of 8) to TRH and age-related normal responses of GH to GRH (4 of 8), LH (6 of 8), and FSH (6 of 8) to GnRH. These findings suggest that long term reserpine administration in combination with a conventional dose of pituitary irradiation is useful in the treatment of Cushing's disease.


Typing for major histocompatibility complex class II antigens in thyroid tissue blocks: association of Hashimoto's thyroiditis with HLA-DQA0301 and DQB0201 alleles.

This study was undertaken 1) to find out whether we can type major histocompatibility class II antigens from the paraffin-embedded series of thyroid tissue, and 2) to investigate whether HLA-DQ genes are involved in conferring a risk of Hashimoto's thyroiditis. To this end we used the polymerase chain reaction to amplify DNA from paraffin-embedded thyroid tissue blocks of histologically proven Hashimoto's disease. We used 46 specimens for HLA-DQA and 32 for DQB typing. The alleles were identified by sequence-specific oligonucleotide hybridizations. Fifty controls from the same geographic region were also typed using peripheral leukocyte DNA. HLA-DQA0301 (in linkage disequilibrium with DR4) was significantly increased (58.7% vs. 32% in controls; chi 2 = 6.73; P less than 0.01) in patients compared to controls. DQB0201 (in linkage disequilibrium with DR3) was also increased in the patient group (66% vs. 36% in controls; chi 2 = 6.63; P less than 0.01). Although DQA0301/DQB0201 heterozygotes (18.8%) were increased in patients compared to controls (6%), the difference was not significant. However, 81% of the patients (26 of 32) were DQA0301 and/or DQB0201 positive compared to 48% of controls (chi 2 5.98; P less than 0.05). We conclude that it is feasible to type HLA antigens from tissue blocks and that susceptibility to Hashimoto's disease is probably mediated through two pathways: DQA0301/DR4 and DQB0201/DR3.


Interleukin-1 is induced in the human placenta by endotoxin and isolation procedures for trophoblasts.

Although freshly obtained placenta contains little or no interleukin-1 (IL-1) mRNA, placenta and isolated trophoblast have been reported to produce significant quantities of bioactive IL-1 in vitro. The present study was designed to determine if endotoxin, a common contaminant of culture medium, and trophoblast isolation procedures could induce IL-1 expression in the placenta. Tissue-extractable IL-1 alpha and IL-1 beta immunoreactive proteins were readily detected in fresh placental membranes, but not placental villi. As little as 10 ng/mL endotoxin were found to induce the expression of IL-1 alpha and IL-1 beta mRNA in intact placental villi cultured in vitro. Intact placenta cultured in the presence of 1.0 microgram/mL endotoxin demonstrated expression of IL-1 alpha and IL-1 beta mRNA and cumulative production and release of immunoreactive IL-1 alpha and IL-1 beta into the medium during 24 h of culture. Placenta incubated in endotoxin-free medium, however, exhibited no detectable IL-1 alpha or IL-1 beta mRNA expression and little or no release of IL-1 alpha or IL-1 beta immunoreactive protein into the medium. When trophoblast cells were freshly isolated by enzymatic digestion, followed by Percoll separation at reduced temperatures to inhibit cell activation, no IL-1 alpha or IL-1 beta mRNA expression was initially detectable. However, IL-1 alpha and IL-1 beta mRNA in isolated trophoblast cells were induced after 30 min of culture in endotoxin-free medium, with maximal induction at 4 h. These results suggest that normally the placenta produces very little, if any, IL-1, and that endotoxin and trophoblast isolation procedures induce IL-1 expression in placental tissues cultured in vitro.


Distribution of F-actin during mouse facial morphogenesis and its perturbation with cytochalasin D using whole embryo culture.

Histological and experimental studies were performed in mouse embryos to elucidate possible roles of actin filaments in the nasal epithelium during facial morphogenesis. C57BL/6 mouse embryos (8.5-11.5 days of gestation) were fixed and frozen sections were stained with rhodamine-phalloidin. Before formation of the nasal placode, there was no specific localization of F-actin. After the nasal placode was formed, intense staining of F-actin was observed at the apical side of the placode. Conversely, it was located at the basal side of the epithelium of developing nasal prominences. By using the whole embryo culture system, perturbation experiments were conducted with cytochalasin D (CD), which inhibits the polymerization of actin filaments. When day-10 embryos were exposed to CD at several concentrations for 24 hr, fusion of nasal prominences was inhibited in a dose-dependent manner. Treatment with a high dose of CD for 2 hr also prevented the same development irreversibly. In contrast, when day-9 embryos were exposed to CD at several concentrations for 24 hr, invagination of the nasal placode was not perturbed at all. The results suggest that apical F-actin plays an essential role in maintaining the close apposition state of the nasal prominences and in the following fusion. During the invagination stage, F-actin might be important in maintaining the epithelial structure, but is not crucial to the initiation of placode invagination.


A morphometric analysis of craniofacial growth in cleft lip and noncleft mice.

Differences in face shape are considered a factor in cleft lip malformation. The purpose of this study was to analyze craniofacial growth in two strains: A/WySn with 28% cleft lip and C57BL/6J without cleft lip. Standardized photographs of 27 A/WySn and 25 C57BL/6J embryos with 34-46 somites (S) were taken in the superior, frontal, and lateral views. Landmarks were located and digitized for computerized analysis of growth change relative to somite number and at stages of face development before, during, and after primary palate closure. The results showed that both strains had similar overall growth patterns with increases in head width and face width, and decreases in nasal pit width. During early palatal closure in C57BL/6J mice, the nasal pit width was unchanged as brain width increased rapidly; and then later, the nasal pit width decreased as brain width increased slowly. However, during early closure in A/WySn mice, the nasal pit width decreased rapidly as brain width increased slowly; and then later, the nasal pit width was unchanged as brain width increased more rapidly. During early palatal closure, the narrower nasal pit width in A/WySn mice appeared to result from delayed growth of the supporting forebrain as the nasal pits become more medially positioned with normal face development. From the lateral view, the maxillary prominence depth was also smaller in the A/WySn strain during early palatal closure. This deficient forward growth of the maxillary prominences and the narrower positioning of the medial nasal prominences in A/WySn embryos appear to reduce the contact between the prominences and thus predispose this strain to cleft lip malformation.


Interleukin-1 beta induces corticotropin-releasing factor-41 release from cultured hypothalamic cells through protein kinase C and cAMP-dependent protein kinase pathways.

Interleukin-1 beta (IL-1 beta) induces a dose-dependent increase in the release of corticotropin-releasing factor-41 (CRF) from dispersed rat fetal hypothalamic cells in culture. This release of CRF could be inhibited by the protein kinase C inhibitor H-7, and by the protein kinase A inhibitor IP-20. This suggests that both protein kinase C and protein kinase A-dependent pathways are involved in the response of CRF to IL-1 beta. Dexamethasone also blocked the CRF response to IL-1 beta, indicating that activated glucocorticoid receptors can inhibit the response of CRF to IL-1 beta.


Ovalbumin is more immunogenic when introduced into brain or cerebrospinal fluid than into extracerebral sites.

The magnitudes of serum antibody responses to ovalbumin have been compared following immunization via cerebral or extracerebral sites in Sprague-Dawley rats. In central nervous system (CNS)-immunized rats, conditions were designed to ensure normal brain barrier permeability. Extracerebral immunization was via the footpad or along pathways of antigen outflow from the CNS. The relative immunogenicity of different injection sites is: CSF greater than brain tissue greater than extracerebral sites. Enhancement of the antibody response to CNS-administered antigen appears to depend on events initiated within the CNS, since ovalbumin injected into blood (which reaches the spleen) or nasal submucosa (which drains to cervical nodes) fails to elicit a similar response.


Scoring mitotic activity in longitudinal sections of mouse embryonic incisors: significant differences exist for labial and lingual inner dental epithelia.

The incisors of rodents (e.g., mice and rats) have an enamel-free lingual surface. It is not known how position specific differential evolution of labial versus lingual inner dental epithelium (IDE) is regulated. The patterns of mitotic activity of lingual and labial preodontoblasts and IDE were determined on sagittal sections of embryonic mouse incisors. No statistical differences were observed for the preodontoblasts. On the other hand the lingual IDE withdraw from the cell cycle before the corresponding labial cells. The incapacity of lingual IDE to differentiate into functional ameloblasts might be a consequence of a failure of cell kinetic dependent acquisition of competence to respond to specific epigenetic control mechanisms.


Palate teratogenicity and embryotoxicity of cyclosporin A in mice.

Mice of the A/J and B10.A/SgSnJ strains were treated with 50 mg of cyclosporin A (CsA) per kg of body weight on day 12 of gestation. There were significantly more fetal resorptions in both the A/J and B10.A strain when treated with CsA than in controls treated with olive oil. A low frequency (7.6%) of isolated cleft palates were induced in the A/J strain, which was significantly greater than that observed in A/J mice treated with olive oil alone. No cleft palates were induced in B10.A, which suggests that any increase in susceptibility that was observed could not be attributed to H-2 linked genes.


Cavitation defects on the lingual ramus: a further expression of Stafne's defect.

Seven archaeological examples of 6,700 mandibles examined were found to have developmental cortical defects on the ramus. The defects, unusual in their position, were grossly and radiographically similar to developmental bone cavities (Stafne's) located in the sublingual region. Of the seven ramus defects, all of which were in adult males, six were in Alaskan Eskimos from Kodiak Island (N = 5) and Nelson Island (N = 1), and one was in a native American from Arkansas. Only five adult males from Finland have been reported with similar defects. A statistical examination of the metrics defining mandibular cavitation defects, along with prevalence data, suggest that the seemingly rare findings of ramus defects are an extreme manifestation of the continuum of severity of the Stafne's defect. Bilateral Stafne's defects also represent a severe manifestation.


Evidence for activation of the complement cascade in the hypoglossal nucleus following peripheral nerve injury.

Following hypoglossal nerve transection in adult rats, immunoreactivity for complement factor C3 and one of its degradation products C3d as well as C4d and immunoglobulin G (IgG) was observed in the ipsilateral hypoglossal nucleus. Double-labelling experiments indicated that these antigens were present in perineuronally located reactive microglial cells. In addition, increased levels of complement factor C3-mRNA was found in perineuronally located cells ipsilateral to nerve lesion. These results suggest that the complement cascade is locally activated in the vicinity of axotomized neuronal perikarya and that microglial cells have a key role in this process, alternatively that C3, C3d, C4d and IgG are involved in other so far unknown processes.


A craniometric study of bicoronal craniosynostosis (BCC).

We compared 20 cases of bicoronal craniosyntosis (BCC) using the linear and angular values of the normal statistical material for the same age and sex. The skull in BCC is significantly shortened and flattened but with normal width. The height of the frontal arch (FNBR) is significantly decreased. The base of the skull is depressed with angle AB2 significantly increased, and angle NTNO significantly decreased and, even in some cases, with negative angular values. This is inducing a sinking of LEA (length from N to T), and in some cases LEA is lower than the line LNO (distance from N to O). The cephalic index (CI) was significantly increased, and the calculated cranial capacity (CC) was significantly decreased. Bregma (BR) is moving anteriorly in BCC, inducing an anterior movement of the calvaria with a bulge around BR. In this anterior movement we designate lack of positive occipital rotation. In BCC there is an evident lack of positive occipital rotation. Superimposing our drawings on the reference line NBA, we found for the first time a movement of anatomical landmarks similar to that observed in the negative occipital rotation in vestibular orientation.


Somatosensory evoked potential testing in infants and children.

Somatosensory evoked potentials (SSEPs) provide the means to assess noninvasively the peripheral and central nervous system in infants and young children. As experienced clinicians know, this is the very system most difficult to adequately evaluate in young children. Physical examination is difficult. SSEPs provide information about function of somatosensory systems not otherwise available. In addition to pathologic states, maturation and sleep produce changes in SSEPs that require careful analysis.


Intraoperative electrophysiological monitoring in children.

Children, like adults, are at risk for neurological injury during a variety of surgical procedures. Intraoperative electrophysiological monitoring (IOM) provides information about the functional integrity of the nervous system during surgery. This information may determine the mechanism of injury and prevent damage by identifying nerves and detecting dysfunction at a reversible stage. Electrophysiological techniques may also help direct therapy by improving injury localization, detect the presence or absence of axonal integrity in peripheral nerve lesions, and identify rootlets with the greatest contribution to spasticity in patients undergoing selective dorsal rhizotomy (SDR). Electrophysiological techniques used are modified from those employed in the diagnostic laboratory. The first portion of this article reviews IOM experience at the Mayo Clinic in patients under 18 years of age from 1985 to 1991. The types of procedures monitored, the monitoring modalities used, technical problems unique to children, and neurological outcome are discussed. Subsequently presented are the application and techniques of electrophysiological monitoring during SDR as currently practiced at the University of Kentucky and other medical centers.