Creation of an isogenic P1-deficient mutant of Haemophilus influenzae biogroup aegyptius

The oligosaccharide chains of pituitary glycoprotein hormones such as human thyroid-stimulating hormone (hTSH) have been shown to be important in biosynthesis, subunit association, secretion and bioactivity. However, the exact biological significance of these glycosylation variants (isoforms) remains controversial. The aim of this paper is to investigate the role of hTSH glycosylation variants in signal transduction. Human pituitary standard TSH (2nd International Reference Preparation 80/558; IRP-hTSH) was treated with neuraminidase, fractionated by isoelectric focusing (IEF) and affinity chromatography using the lectins concanavalin A (Con A) and lentil. To determine the in vitro bioactivity of these hTSH isoforms, simultaneous measurement of cAMP formation and inositol phosphates release was applied in two different cell systems (CHO cells stably and Cos-7 cells transiently transfected with hTSHR cDNA). Desialylated TSH variants showed a significantly increased ratio of bioactivity to immunoreactivity for cAMP production in CHO-R cells (B/I ratio desialylated variants: 3.54 +/- 0.005; B/I ratio sialylated variants: 2.84 +/- 0.01 P < 0.05). Testing the bioactivity of hTSH glycosylation variants isolated by IEF, we found basic variants to be significantly more active than acidic ones in stimulating the cAMP formation in CHO-R cells (B/I ratio basic variants: 9.92 +/- 0.64; neutral variants: 5.98 +/- 0.07; acidic variants: 2.80 +/- 0.12; P < 0.01). There were no differences in stimulation of IP-release. High-mannose TSH variants (firmly bound to Con A) showed greater potency to stimulate cAMP formation and IP-release in both CHO-R and Cos-7 cells than biantennary TSH variants (weakly bound to Con A). Both core-fucosylated (lentil-bound) and core-unfucosylated (lentil-unbound) TSH variants proved to be strong stimulators of cAMP release in CHO and Cos-7 cells. In CHO-R (Cos-7) cells, 400 microU/ml core-fucosylated TSH stimulated cAMP formation 14(2.6)-fold, core-unfucosylated TSH 7.3(2.3)-fold over control values. In contrast to our findings of cAMP activation by both core-fucosylated and core-unfucosylated TSH variants, release of IPs was stimulated only by, core-fucosylated (lentil-bound) TSH variants and not by TSH variants lacking core-fucose residues (lentil-unbound TSH). This was true for both CHO-R and Cos-7 cells. The lentil-unbound TSH therefore showed an identical differential activation of signal transduction pathways in two different cell systems: strong stimulation of the cAMP-cascade without activation of IPs release (P < 0.05). In conclusion, we showed for the first time for TSH that the two dominant intracellular signal transduction systems (cAMP formation and IPs release) are activated to different degrees by hTSH glycosylation variants.     

Changes in the degree of sialylation of carbohydrate chains modify the biological properties of circulating thyrotropin isoforms in various physiological and pathological states

Variation in asparagine-linked carbohydrate chains have a major impact on TSH biological properties. In particular, highly sialylated TSH is characterized by impaired intrinsic bioactivity and prolonged half-life. The aim of the present study was to investigate the changes in the degree of sialylation of circulating TSH isoforms that may occur in several physiological and clinical situations. Bioactivity and terminal sugar residues of immunopurified TSH were studied in 26 normal adults (day- and nighttime serum pools), 2 cord serum pools from normal fetuses during the third trimester, 1 fetus with primary hypothyroidism (PH; 27th week), 1 fetus with resistance to thyroid hormone (RTH; 28th and 33rd weeks), 24 patients with PH (before and during L-T4 treatment), and 5 patients with RTH before and during triiodothyrocetic acid (TRIAC) treatment. Nighttime TSH isoforms have an increased degree of sialylation compared to daytime TSH (35.8 +/- 9.7% vs. 23.8 +/- 5.8%; P < 0.03), thus accounting for the lower bioactivity [biological/immunological TSH ratio (TSH B/I), 1.3 +/- 0.4 vs. 2.0 +/- 0.2; P < 0.0007]. In adult PH, TSH isoforms are highly sialylated (45.4 +/- 7.6%; P < 0.007), showing an impaired bioactivity (0.7 +/- 0.3; P < 0.001). L-T4 therapy was accompanied by a trend toward normalization of TSH biological properties; TSH B/I was higher (1.0 +/- 0.3; P < 0.01), and the degree of sialylation was lower (36.8 +/- 7.0%; P < 0.02). A significant inverse correlation between TSH B/I values and the degree of sialylation was observed (P < 0.001). In normal fetuses, extremely bioactive asialo-TSH isoforms are circulating during the 3rd trimester. The impaired thyroid hormone action, such as that occurring in hypothyroid or RTH fetuses, induces an early expression of alpha-2,6-sialyltransferase activity within thyrotropes and results in the secretion of high amounts of sialylated TSH isoforms (34.6% and 26.3%). A hybrid TSH with peculiar terminal sugar residues and enhanced bioactivity is circulating in patients with RTH (TSH B/I, > or = 2.2). Treatment with low doses of TRIAC can initially reduce thyroid hormone secretion in RTH, mainly through the secretion of TSH isoforms with changed terminal sugar residues and reduced bioactivity (TSH B/I, 0.9-1.7). In conclusion, changes in the terminal sialic acid residues modulate the biological properties of circulating TSH, play a relevant physiopathological role in various situations, and contribute to adjust thyroid-stimulating activity to temporary needs.     

Preimplantation genetic diagnosis of DiGeorge syndrome

By combining evolutionary considerations, sequence comparisons and homology modeling we have designed recombinant human thyroid-stimulating hormone (hTSH) analogs with increased receptor binding and activity. The introduction of seven basic residues into the peripheral loops of hTSH resulted in up to a 50,000-fold increase in receptor binding affinity and 1300-fold increase in intrinsic activity. Such analogs are not only of potential clinical interest but can be tools to explore molecular aspects of conventional as well as nonclassical actions of glycoprotein hormones. These design strategies should be applicable to the development of novel analogs of other related hormones and growth factors with a variety of therapeutic and basic science applications, particularly for proteins that have undergone evolutionary decrease in bioactivity.     

Disulfide bonds 7-31 and 59-87 of the alpha-subunit play a different role in assembly of human chorionic gonadotropin and lutropin

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consists of cystine knot structure. We previously eliminated the disulfide bonds 7-31 and 59-87 in alpha-subunit without significantly affecting assembly with human CG beta-subunit. To study the role of these disulfide bonds in dimerization with other beta-subunits, the wild-type or mutated alpha gene was contransfected with the wild-type human LH beta or FSH beta gene into Chinese hamster ovary (CHO) cells or GH3 cells, and assembly was assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or-beta serum, and SDS-PAGE. Our data revealed that disruption of either disulfide bond 7-31 or 59-87 in the alpha-subunit markedly reduced the dimer formation with LH beta-subunit in both CHO and GH3 cells, whereas it did not significantly affect the assembly of FSH. This suggests that the regions in the alpha-subunit recognized by beta-subunits for assembly are different among gonadotropins.     

Aspartate-474 in the first exoplasmic loop of the thyrotropin receptor is crucial for receptor activation

Asp-474 in the first exoplasmic loop of the thyrotropin receptor (TSHR), which is conserved among all glycoprotein hormone receptors, was mutated to Glu which is similarly charged but is longer by one methylene group and expressed in Cos-7 cells. Cells expressing this mutant receptor showed markedly impaired TSH- and TSAb (thyroid stimulating antibody)-stimulated cAMP responses with no effect on TSH binding affinity when compared with cells expressing a similar number of wild-type receptors. These results suggest the importance of Asp-474 in TSHR in receptor activation as demonstrated for LHR (lutropin receptor), but this, unlike LHR, is not due to the electrostatic interaction of this Asp residue with the alpha-subunit Lys-91 of the hormone.     

The human follitropin alpha-subunit C terminus collaborates with a beta-subunit cystine noose and an alpha-subunit loop to assemble a receptor-binding domain competent for signal transduction

FSH is a member of the pituitary/placental glycoprotein hormone family including luteinizing hormone, thyroid-stimulating hormone, and chorionic gonadotropin. These heterodimeric hormones share a common alpha-subunit and a highly homologous but distinct beta-subunit. The determinant loop of the FSH beta-subunit acts both as a specificity discriminator and as an essential receptor-binding site. The three-dimensional structure of hCG illustrates the proximity of the determinant loop to the carboxyl-terminal residues of the common alpha-subunit. Thus, site-directed mutagenesis was used to mak high-resolution substitutions at this carboxyl-terminal locus. The effects of those substitutions were studied. Twelve single mutations and one composite mutation were made of the region of hFSH alpha 74-92, each residue substituted by alanine. Side chain replacement in this region of FSH proved to be detrimental to binding. hFSH with mutations of either alpha S85A, alpha T86A, alpha K91A, or alpha S92A only retained 10% or less of the hFSH receptor-binding activity, while compared to these, mutants alpha H79A, alpha Y88A, and alpha Y89A retained slightly more binding activity. The single mutant alpha F74A and composite mutant alpha V76A/E77A binding activity was reduced to half of that of wild-type (WT) hFSH. In contrast, mutations of either alpha K75A, alpha T80A, alpha H83A, or alpha H90A did not adversely affect receptor binding, demonstrating the specificity of observed effects. The hFSH and mutant hormones were tested in an in vitro bioassay for stimulation of progesterone production. Those mutations that did not affect receptor binding (alpha K75A, alpha T80A, alpha H83A, and alpha H90A) did not affect signal transduction, measured by progesterone responses. After comparison of wild-type and mutant hFSH activities determined in radioreceptor assays (ID50) and in vitro bioassays (ED50), it became evident that signal transduction correlated with receptor binding.     

Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles

Using a highly sensitive bioassay for TSH, in which human thyroid follicles incorporate 125I and release de novo synthesized thyroid hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human thyroid follicles is too weak to cause hyperthyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperthyroidism when produced excessively.     

Criteria of cure and follow-up of central hyperthyroidism due to thyrotropin-secreting pituitary adenomas

The recorded number of patients with central hyperthyroidism due to TSH-secreting pituitary adenoma doubled in the last few years after the introduction of ultrasensitive TSH assays in the assessment of thyroid function; however, information about the results and the criteria for cure after pituitary surgery is scanty. Seventeen patients with a TSH-secreting adenoma, diagnosed on the basis of detectable TSH levels in the face of high free thyroid hormone concentrations and pituitary lesion at neuroimaging, underwent pituitary surgery. Hypersecretion of other pituitary hormones was diagnosed in 5 of 17 patients. Four patients were initially misdiagnosed and treated with thyroid surgery or radioiodine therapy. The majority (86%) of hyperthyroid patients normalized thyroid hormone concentrations and regained euthyroidism, although pituitary imaging, alpha-subunit, and alpha-subunit/TSH molar ratio normalized in only 47%, 54%, and 58% of patients, respectively. Moreover, TSH secretion was normally suppressed by T3 in 40% of the patients. Interestingly, the finding of undetectable TSH levels 7 days after surgery was highly predictive of successful outcome. During long term follow-up, there was one relapse of hyperthyroidism. Early diagnosis of TSH-secreting adenomas permits a high rate of remission of hyperthyroidism after surgery. However, normalization of thyroid function alone does not necessarily reflect complete removal of the tumor, and more comprehensive criteria of cure based on pituitary imaging, hormone measurement, and suppression of TSH during T3 administration should be used. Lastly, all patients need an accurate long term follow-up to monitor the possible recurrence of the adenoma.     

Changes in circulating levels of LH, FSH, LHbeta- and alpha-subunit after gonadotropin-releasing hormone, and of TSH, LHbeta- and alpha-subunit after thyrotropin-releasing hormone

The effect of the gonadotropin-releasing hormone (LRH) and thyrotropin-releasing hormone (TRH) on the blood levels of LH, FSH and TSH, and LHbeta- and alpha-subunit have been studied in 4 normal subjects during the first 20 min after administration of these releasing hormones. Increases in serum immunoreactive LH, LHbeta and alpha-subunit were seen in all subjects after LRH (100 mug iv) but in all subjects the rise in LH was preceded by a rise in alpha-subunit. All subjects showed an increase in TSH and 3 of the 4 subjects a rise in alpha-subunit after TRH (200 mug) but the alpha-subunit responses were smaller and less consistent than after LRH. Levels of LHbeta remained unchanged after TRH. The results demonstrate that the immunoreactive alpha-subunit of the pituitary glycoprotein hormones can be released independently of the intact hormones and that release occurs in response to the same releasing hormones, LRH and TRH, that release the intact hormones.     

The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase

In the structure of Escherichia coli aspartate transcarbamoylase with CTP bound [Kosman, R. P., Gouaux, J. E., and Lipscomb, W. N. (1993) Proteins, Struct. Funct. Genet. 15, 147-177] Lys-6 and Glu-62 form a salt-link between two regulatory chains. However, recent X-ray structural studies suggest that side chain and backbone interactions existed between Glu-62 and Ile-12. Thus the interaction between Glu-62 and Ile-12 may help to establish the correct conformation of the nucleotide binding site. The present study of two single-site mutant enzymes, Glu-62r-->Ala and Ile-12r-->Ala, was undertaken to investigate whether the role of Glu-62 is to maintain the stability of the interface between the regulatory chains in the dimer, or interacts with the side chain of Ile-12r to define the nucleotide binding site. For both the mutant enzymes, the maximal velocity, the aspartate saturation at half the maximal velocity, and Hill coefficient were close to wild-type values. The Glu-62r-->Ala enzyme showed enhanced regulatory effects with ATP, CTP, and UTP. As a result of this mutation the enzyme losses its ability to discriminate between CTP and UTP. For the Ile-12r-->Ala enzyme, the heterotropic properties were reduced or eliminated. The enhanced regulatory effects observed with the Glu-62r-->Ala enzyme do not seem to be consistent with the presence of a salt-link between Glu-62r and Lys-6r. However, based upon kinetic data of the unique but completely opposite heterotropic properties of the two mutant enzymes, it is suggested that the side chain interaction between Glu-62r and Ile-12r helps to define the conformation of the effector binding pocket. In this study, we report the properties of both the Glu-62r-->Ala and Ile-12r-->Ala enzymes and their importance for the heterotropic activation and inhibition of aspartate transcarbamoylase.     

Thyrotropic action of human chorionic gonadotropin

Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent with the finding that the beta-CTP truncated hCG with higher thyrotropic potency is substantially deglycosylated and desialylated in the beta-subunit relative to intact hCG because all four O-linked glycosylation sites occur within the missing C-terminal extension. The desialylated hCG variant also interacts directly with recombinant hTSH receptors transfected into human thyroid cancer cells. There is thyroid-stimulating activity in sera of normal pregnant women, and this correlates with serum hCG levels. The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, perhaps being about 1 mU/L less than nongravid levels, but not high enough to induce overt hyperthyroidism. Maternal thyroid glands may secrete more thyroid hormone during early pregnancy in response to the thyrotropic activity of hCG that overrides the normal operation of the hypothalamic-pituitary-thyroid feedback system. Biochemical hyperthyroidism associated with hyperemesis gravidarum has been attributed to hCG. In patients with hyperemesis gravidarum, thyrotropic in serum correlated with hCG immunoreactivity, and the severity of vomiting as indicated by clinical and biochemical parameters correlated with the degree of thyroid stimulation. To understand the thyrotropic action of hCG, it is necessary to know whether hCG activates the same domain of the TSH receptor as does TSH. The identification of the molecular structure of the hCG isoform with the highest thyrotropic potency will resolve the enigma of gestational thyrotoxicosis and the hyperthyroidism associated with trophoblastic disease and hCG-producing tumors.     

Single chain dimers of MASH-1 bind DNA with enhanced affinity

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.     

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.     

Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules

A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z-DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z-DNA binding clones, but they bound B-DNA and denatured DNA as well as Z-DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.     

A rare case of a thyrotropin-secreting pituitary tumor which responded to methimazole

A 52-year-old Japanese male complained of palpitations and excessive sweating. He showed evidence of hyperthyroidism, but without suppression of the serum thyroid-stimulating hormone (TSH). On admission, the serum level of the alpha-subunit of TSH was elevated, but tests for thyroid autoantibodies were negative. The TSH response to thyrotropin-releasing hormone (TRH) was blunted. Imaging studies revealed a pituitary tumor 2 cm in diameter. Administration of methimazole lowered the serum levels of thyroid hormones, and elevated the serum level of TSH. These findings led to the diagnosis of a thyrotropin-secreting pituitary tumor. Immunohistochemical analysis and electron microscopic findings verified the diagnosis.     

Mono- and dysfunctional nitrogen mustard analogues of the DNA minor groove binder pibenzimol. Synthesis, cytotoxicity and interaction with DNA

Two series of mono- and dysfunctional aniline mustards linked to a bisbenzimidazole minor groove binder have been prepared using a new method (polyphosphate ester-mediated direct coupling of appropriate mustard acids with a preformed advanced phenylenediamine intermediate). As the linker chain attaching the mustard was lengthened the binding site size of the compounds to calf thymus DNA remained essentially constant at 2.6 nucleotides, but reversible binding strength declined by a factor of 2. Analogues with longer linker chains alkylated DNA much more rapidly than those with shorter chains, consistent with the electronic factors. The short chain analogues also failed to alkylate a 120 bp HindIII to Bg/II fragment of the gpt gene, as measured by gel electrophoresis cleavage assays. The longer chain analogues (both mono- and dysfunctional mustards) showed patterns of DNA alkylation that varied with chain length. In particular, while most compounds showed substantial N7 alkylation at many guanine residues, the analogue with a (CH2)3 linker chain showed strong alkylation at adenine sites in poly-AT regions. For the longer chain analogues, the bifunctional mustards were substantially (10- to 20-fold) more cytotoxic than the corresponding monofunctional analogues.     

Central hyperthyroidism

Central hyperthyroidism is a rare condition in which thyrotoxicosis results from primary overproduction of TSH by the pituitary gland with subsequent thyroid enlargement and hyperfunction. The two known causes of central hyperthyroidism are TSH-producing pituitary tumors (TSHomas) and the syndrome of PRTH. Both of these entities are characterized by clinical thyrotoxicosis, diffuse goiters, elevated circulating levels of free T4 and T3, and a nonsuppressed serum TSH. It is critical to distinguish central hyperthyroidism from the much more common types of primary hyperthyroidism, all of which have undetectable TSH values. TSHomas and PRTH can usually be differentiated from one another by measuring the serum alpha-subunit and the TSH response to intravenous TRH or exogenous thyroid hormone, and by pituitary imaging studies. TSHomas are usually benign adenomas arising from the monoclonal expansion of neoplastic thyrotropes. Causative oncogenes have not yet been convincingly identified. PRTH is a nonneoplastic disorder caused by inherited mutations in the gene for the thyroid hormone receptor beta; it is a poorly understood variant of GRTH. For unclear reasons, in PRTH, the pituitary gland is resistant to the feedback inhibitory effects of circulating thyroid hormones while peripheral tissues respond normally, causing patients to experience the toxic peripheral effects of thyroid hormone excess. TSHomas are best treated by transphenoidal surgical removal. Radiotherapy is indicated for inoperable or incompletely resected tumors. Octreotide administration is a useful adjunct for preoperatively reducing tumor size and for the medical management of surgical treatment failures. PRTH is ideally treated by chronically suppressing TSH secretion with medications such as D-thyroxine, TRIAC, octreotide, or bromocriptine. If such therapy is ineffective or unavailable, thyroid ablation with radioiodine or surgery may be employed with subsequent close monitoring of both thyroid hormone status and pituitary gland size.     

Glycoprotein hormone alpha-subunit secretion in non-functioning pituitary adenomas

The aim of the present study was to investigate the prevalence of elevated free glycoprotein hormone alpha-subunit in different pituitary adenomas, to establish the diagnostic value of the basal and stimulated free alpha-subunit secretion in non-functioning adenomas. Serum basal levels of alpha-subunit were increased in 1 of 22 untreated, in 1 of 16 operated patients with non-functioning adenoma, in 6 of 28 untreated, in 1 of 7 operated patients with acromegaly, in 0 of 5 untreated prolactinomas and in 0 of 1 untreated gonadotrop adenoma. Overall free alpha-subunit levels were increased in 9 of 79 cases (11.4%). In 6 of 9 patients with untreated non-functioning adenoma thyrotrop hormone releasing hormone caused an abnormal--paradox--elevation of serum alpha-subunit. These data indicate that measurement of basal and stimulated alpha-subunit is of relatively poor value in the diagnosis of non-functioning pituitary adenomas. The transsphenoidal surgery did not resulted in a change of alpha-subunit secretion neither in patients with non-functioning adenoma nor with acromegaly. The present data confirm the view that non-functioning pituitary adenomas are not homogeneous since this subset of tumors includes adenomas that either do not secrete measurable amounts of free alpha-subunit or produce normal or supranormal amounts of subunits as consequence of still undefined biosynthetic abnormalities.     

Discrepancies between thyrotropin (TSH) measurement by four sensitive immunometric assays

A comparison of four different commercial immunometric thyrotropin (TSH) assays (Amerlite R TSH-30 Ultrasensitive assay from Kodak, BeriLux R hTSH from Behring Werke, Delfia R hTSH Ultra from Wallac and IMX R Ultrasensitive hTSH from Abbott) was made by measuring serum TSH in 81 consecutive patients referred to hospital for various reasons with a serum TSH less than 0.8 mlU/l in the IMX assay. The analytical and functional assay sensitivities of each of the assays were analysed. Even though three of the methods had a sensitivity corresponding to third generation assays, we could only demonstrate an overall agreement of serum TSH when comparing two of the kits. The measurements in Delfia Ultra and Berilux showed good agreement (P = 0.7, paired t-test and bias = 0.003 mIU/l), while the comparisons between the other assays showed different measurements (P < 0.00001, paired t-test and bias more than 0.07 mIU/l). Differences in the calibrators used in the assays might explain some of the discrepancy, although all methods were calibrated according to the same international standard. Also, differences in the specificity of the TSH monoclonal antibodies used in the assays might be an evident explanation and further studies of the specificity of the monoclonal antibodies are needed. An international collaborative study to clarify reasons for the differences between the TSH assays and to standardize the measurements is recommended.     

Thyrotropic deficiency

Central hypothyroidism (thyrotropic deficiency) is due to a defect in TSH secretion by thyrotrophs (or alternatively to an altered bioactivity of TSH). Central hypothyroidism is rare and is often associated with other pituitary deficiencies as it is generally encountered in case of hypothalamo-pituitary tumoral process. Clinical symptoms are milder than those of primary thyroid failure. Diagnosis is based on free T4 measurement whose level is decreased while TSH concentration is normal or minimally increased, reflecting an alteration in the bioactivity of TSH. Replacement therapy is monitored by T4 level measurement: the objective is to obtain normal T4 levels. TSH concentration must not be taken into account for the adjustment of the thyroxine doses.