The W520X mutation in the TSHR gene brings on subclinical hypothyroidism through an haploinsufficiency mechanism.

BACKGROUND: TSHR is a G-protein-coupled seven transmembrane domain receptor that activates the two major signal transduction pathways: the Gαs/adenylate cyclase and the Gαq/11/phospholipase C pathways. Inactivating mutations in the TSHR gene have been demonstrated to be responsible for subclinical hypothyroidism, a disorder characterized by elevated serum TSH concentrations despite normal thyroid hormones levels. AIM: We identified in a child a nonsense mutation (W520X) in the third transmembrane domain of the TSHR that causes the lack of the C-terminus portion of the receptor. The functional significance of this variation was assessed in vitro. MATERIAL/SUBJECT AND METHODS: The W520X mutation was introduced into the pSVL vector containing the wild-type sequence of TSHR gene. Wild-type and mutated vectors were expressed in Chinese Hamster Ovary (CHO) cells, and cAMP, inositol phosphate (IP), immunofluorescence and FACS analyses were performed. RESULTS: Transfection with pSVL-TSHR vector induced basal cAMP and IP production in the absence of TSH stimulation, indicating a constitutive activity for the TSHR. An impairment of receptor function was demonstrated by the observation that cells expressing the mutant TSHR exhibited a lower second messenger production with respect to the wild-type, despite a normal expression of the receptor at the cell surface. CONCLUSIONS: The mechanism through which the W520X mutation exerts its effect is more likely haploinsufficiency rather than a dominant-negative effect. This could explain the phenotype of our patient, who has a hormonal pattern in the range of a mild subclinical hypothyroidism, without an overt disease phenotype.

Intravenous fluid use in the acutely unwell adult medical inpatient: improving practice through a clinical audit process.

BACKGROUND: Our Trust developed a clinical guideline to improve the prescribing and use of intravenous (IV) fluids based on the British consensus guidelines on IV fluid therapy for adult surgical patients. We audited the effect of targeted interventions to improve performance against this guideline. METHOD: There were 53 IV fluid prescription charts in the pre-intervention audit and 48 in the post-intervention audit. Data was collected on the seven local practice standards ('local gold standards') in the clinical guideline; compliance with all of them was necessary to meet the IV fluid prescribing bundle of care. RESULTS: The proportion of prescriptions which met the IV fluid prescribing bundle of care increased (3.8% to 22.9% [p=0.004]) and the legibility of the IV fluid prescription increased (28.3% to 56.3% [p=0.004]). CONCLUSION: We have shown that the process of prescribing, administering and monitoring IV fluid use can be significantly improved through a range of targeted multi-disciplinary interventions.

Acylated/unacylated ghrelin ratio in cord blood: correlation with anthropometric and metabolic parameters and pediatric lifespan comparison.

CONTEXT: Ghrelin is a peptide with multiple functions that circulates in acylated (AG) and unacylated (UAG) forms. However, the role of ghrelin in neonates (NN) remains to be clarified. OBJECTIVE: The aim of this study was to determine ghrelin concentrations of the two forms in NN to clarify their biological roles. As such, ghrelin levels at birth were compared with those in later life. SETTING AND DESIGN: Tertiary Care Center. In this cross-sectional study, we evaluated AG, UAG, AG/UAG ratio, and insulin levels in venous cord blood from NN and in fasted normal weight (NW) and obese (OB) children, both prepubertal and pubertal. SUBJECTS: We studied 82 NN, 82 NW, and 58 OB children. RESULTS: AG levels were lower in NN than in NW and OB children (P<0.0001), more specifically the prepubertal NW and OB children (P<0.0001). UAG levels were higher in NN than in NW and OB children (P<0.0001). Therefore, the AG/UAG ratio was lower in NN than in NW and OB children (P<0.0001). NN showed insulin levels similar to NW and lower than OB children (P<0.0001). At birth UAG was positively correlated with AG (Pearson: 0.425; P<0.0001) and negatively with insulin (-0.253; P<0.02). In NW and OB, UAG and AG were positively correlated to each other and negatively correlated with insulin and body mass index (-0.566; P<0.0001). CONCLUSIONS: NN compared with children, showed higher UAG and lower AG levels. The AG/UAG ratio showed a very different profile in NN, being lower than in NW and OB children, thus suggesting a different metabolic function for the two forms in NN. Further studies are needed to clarify the exact role of the different ghrelin forms in NN.

Lack of association between the tetranucleotide repeat polymorphism in the 3'-flanking region of the leptin gene and hypertension in severely obese patients.

Conflicting data suggest an association between leptin gene polymorphisms and essential hypertension independently of obesity. The aim of this study was to evaluate, in severely obese subjects, the role of one of these polymorphic markers in relation to the development of hypertension. The study included 325 obese patients with mean body mass index (BMI) of 46+/-6.94 kg/m2. One hundred sixty-six were hypertensive and 159 normotensive. In both groups, the presence of a tetranucleotide repeat in the 3' flanking region of the Ob gene was investigated using polymerase chain reaction (PCR). Due to the genetic variant, in the region studied it is possible to distinguish two alleles with different size distribution: Class I (shorter one) and Class II (longer one). Class I and Class II allele frequencies were not significantly different in obese patients when analyzed according to the presence or absence of hypertension. The results presented herein do not support a significant association of this Ob gene polymorphism with hypertension. These findings are in contrast with that reported in other populations. However, we cannot rule out that different ethnicity and/or phenotypic variability might mask small effects.

Predictors of postabsorptive ghrelin secretion after intake of different macronutrients.

CONTEXT: Release of ghrelin, a gastrointestinal hormone regulating feeding and energy balance, is blunted in obesity, a condition associated with insulin resistance. OBJECTIVE: The objective was to identify anthropometric and metabolic predictors of postabsorptive ghrelin secretion. DESIGN: We evaluated ghrelin, insulin, glucose, and leptin secretion overnight and after intake of different macronutrients. SUBJECTS: Ten obese subjects (age, 31.8 +/- 2.5 yr; body mass index, 43.4 +/- 0.8 kg/m(2)) and six lean subjects (age, 33.5 +/- 2.4 yr; body mass index, 21.8 +/- 1.4 kg/m(2)) participated in the study. MAIN OUTCOME MEASURES: The main outcome measures were resting energy expenditure (REE); fat mass; nighttime approximate entropy (ApEn) and synchronicity (cross-ApEn) of ghrelin, insulin, and leptin; insulin sensitivity by homeostatic model approach insulin-sensitivity (HOMA-S%); postabsorptive area under the curve (AUC); and Delta of ghrelin, insulin, glucose, and leptin after carbohydrate-, lipid-, and protein-rich test meals. RESULTS: Nighttime ApEn scores were higher in obese than lean subjects (P < 0.01). Cross-ApEn revealed a synchronicity between ghrelin-insulin, ghrelin-leptin, and insulin-leptin in both groups. Compared with baseline, ghrelin decreased significantly (P < 0.01) in lean and obese subjects after carbohydrates (42.2 vs. 28.5%; P < 0.05), lipids (40.2 vs. 26.2%; P < 0.01), and proteins (42.2 vs. 26.3%; P < 0.01) devoid of between-meal ghrelin differences. Significant associations occurred between nocturnal ghrelin ApEn and insulin (r = 0.53; P < 0.05), postmeal ghrelin AUCs and REE (r = -0.57; P < 0.05), and HOMA-S% (r = 0.52; P < 0.05), postmeal ghrelin Delta and HOMA-S% (r = 0.60; P < 0.05). REE (beta = -0.57; P = 0.02) and ghrelin ApEn (beta = -0.62; P = 0.01) were predictors of postmeal ghrelin AUC and Delta, respectively. CONCLUSIONS: Obesity determined a decreased orderliness of ghrelin secretion and a relative loss of ghrelin-insulin synchrony. Postabsorptive ghrelin secretion decreased significantly both in obese and lean subjects, was related to insulin sensitivity, and was predicted by energy expenditure and hormone pulsatility.

Neuroendocrine-like differentiation of non-small cell lung carcinoma cells: regulation by cAMP and the interaction of mac25/IGFBP-rP1 and 25.1.

The need to develop more effective therapies for lung cancer has led to investigations in understanding the molecular mechanisms of the differentiation process, in particular neuroendocrine (NE) differentiation. Recent studies have demonstrated that NE differentiation in non-small cell lung carcinoma (NSCLC) is not uncommon. Those NSCLCs with NE differentiation are considered a form of in transition NE carcinoma and show a more aggressive clinical course compared with NSCLC without NE differentiation. 25.1, a novel protein interacting with mac25/insulin-like growth factor-binding protein-related protein 1 (mac25/IGFBP-rP1), induced NE-like differentiation when collectively overexpressed in M12 prostate cancer cells. We have examined mac25/IGFBP-rP1 and 25.1 as potential molecular regulators in vitro of the NE-differentiation process in lung cancer. In a panel of SCLC and NSCLC cell lines, mac25/IGFBP-rP1 and 25.1 were expressed at higher levels in SCLC. An increase and sustained activation of adenosine 3',5'-cyclic monophosphate (cAMP) levels induced NE-like differentiation in NSCLC cell lines, and a concomitant increase in the expression of mac25/IGFBP-rP1 and 25.1 was observed during the cAMP-regulated differentiation of NCI-H157 cells, suggesting the involvement of these proteins. Furthermore, the collective overexpression of mac25/IGFBP-rP1 and 25.1 in NSCLC cells induced NE-like differentiation as early as 6 h postinfection. The present data suggest that mac25/IGFBP-rP1 and 25.1 may play a functional role in the NE differentiation of NSCLC cell lines and may provide a novel therapeutic target for treating lung cancers, in particular NSCLC with NE differentiation.

Butyrate, a histone deacetylase inhibitor, activates the human IGF binding protein-3 promoter in breast cancer cells: molecular mechanism involves an Sp1/Sp3 multiprotein complex.

Specific cell growth stimulators and inhibitors regulate IGF binding protein-3 (IGFBP-3), where in turn IGFBP-3 mediates their biological effects. The molecular mechanism(s) by which these factors regulate IGFBP-3 are unknown. Sodium butyrate, a histone deacetylase inhibitor causing growth arrest and differentiation, increases IGFBP-3 expression. We investigated the molecular mechanism of this induction using an IGFBP-3 promoter reporter system in MCF-7 and Hs578T breast cancer cells. IGFBP-3 promoter activity was induced up to 40-fold following a 24-h treatment with sodium butyrate and 46-fold in cells treated with trichostatin A, a pure histone deacetylase inhibitor. Deletion analysis of the IGFBP-3 promoter identified key sodium butyrate-responsive element(s) to a 45-bp region containing consensus binding sites for Sp1 and activating protein-2. Sp1 binding to the Sp1 site and Sp3 to the activating protein-2/GA-box played a functional role in sodium butyrate's activation of the IGFBP-3 promoter, however, with no change in binding direct sodium butyrate regulation was attributed to cofactors. The histone acetyltransferase p300 and histone deacetylase-1 were identified in multiprotein complexes containing DNA bound Sp1 and Sp3, with p300 accumulating following sodium butyrate treatment. Taken together, these data suggest that sodium butyrate increases IGFBP-3 expression by activating the IGFBP-3 promoter via an Sp1/Sp3 multiprotein complex, a mechanism that may be important for other key regulators of IGFBP-3.

A conserved region in the first intron of the insulin receptor gene binds nuclear proteins during adipocyte differentiation.

The insulin receptor gene is induced 8 to 10-fold during adipocyte differentiation. Plasmids containing the promoter, exon 1 and a portion of the first intron from either the mouse or human gene are able to modulate the expression of an insulin receptor/CAT gene 3 to 7-fold during differentiation. We have shown that several nuclear proteins from both preadipocyte and adipocyte nuclear extracts bind to two discrete sites within a 278-bp region in the 5' end of the first intron. Sequence comparison between the first intron of the human gene and the mouse gene shows two regions of sequence identity which correspond to the protein binding regions detected by DNase footprinting. One of these sites binds proteins that are enriched in adipocyte nuclear extracts and can be competed by adipose regulatory element, ARE6.

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings.

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.

Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa.

The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.

A catalase from Streptomyces coelicolor A3(2).

Catalase was purified from the Gram-positive bacterium Streptomyces coelicolor A3(2) in a three-step purification procedure comprising (NH4)2SO4 fractionation, Phenyl-Sepharose chromatography and Mono Q chromatography. The purification of catalase, as judged by the final specific activity of 110,000 U mg-1, was 250-fold with a 35% yield. The native protein was a homotetramer with a subunit M(r) 55,000. N-terminal and internal peptide sequence analyses showed that there was a high degree of sequence similarity between the S. coelicolor catalase and other microbial and mammalian catalases. Southern blot analysis indicated that there was a single catalase gene in S. coelicolor. The specific activity of catalase throughout the growth of batch cultures was investigated and elevated catalase activity was found in stationary-phase cells.

Proximal enhancer of the human insulin receptor gene binds the transcription factor Sp1.

The insulin receptor is a growth regulator present on the surface of most cells that transmits a mitogenic signal in response to insulin. Thus, the gene for the insulin receptor is constitutively expressed at low levels in all cells. We characterize a constitutive enhancer element that is present in the proximal promoter of the human insulin receptor gene. We have localized the enhancer to a 26-base-pair (26-bp) sequence from -528 to -503. When this sequence is inserted into the proximal promoter, a three- to fourfold increase in promoter activity is observed, and when two copies are inserted, a five- to sixfold increase is seen. Electrophoretic mobility shift analysis demonstrates that nuclear factors binding to this sequence are found in many different cell types. At least two proteins with different specificities bind within this 26-bp sequence. The identity of the predominant binding protein is Sp1, because an oligonucleotide composed of an Sp1 consensus binding sequence can compete for several of the DNA-protein complexes. In addition, we demonstrate that purified Sp1 can bind to the 26-bp oligonucleotide and that this complex comigrates with a DNA-protein complex formed with a HeLa nuclear extract. Finally, an antibody to human Sp1 protein is able to bind to the enhancer DNA/HeLa protein complex and supershift this complex. These findings suggest that this sequence corresponds to a general element that may contribute to the ubiquitous expression of the human insulin receptor gene.

The amino acid sequence of the small monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe.

The amino acid sequence of the monomeric 2,3-bisphosphoglycerate (BPG)-dependent phosphoglycerate mutase (PGAM) from the fission yeast Schizosaccharomyces pombe has been determined. Amino acid sequencing of proteolytic fragments of the enzyme showed the S. pombe mutase to be similar in sequence to the tetrameric enzyme of baker's yeast (Saccharomyces cerevisiae). An S. pombe cDNA library was screened using a PCR fragment generated from two oligonucleotides complementary to sequences encoding the regions at the two active-site histidine residues. The 0.63 kb cDNA encoded an open reading frame of 210 amino acids. This sequence agreed completely with sequences of peptides derived from the purified protein. The amino acid sequence of S. pombe PGAM is 43% identical with that of S. cerevisiae PGAM and shows an equally high degree of identity with BPG-dependent PGAMs from other sources. However, the sequence of the S. pombe enzyme differs from other BPG-dependent enzymes in three important ways: (i) it does not contain the alanine- and lysine-rich sequence of amino acids at the C-terminus which have been proposed to constitute a flexible tail involved in catalysis; (ii) the sequence spanning residues 122-146 (S. cerevisiae PGAM numbering) is not present in the S. pombe PGAM sequence; in the S. cerevisiae PGAM crystal structure this stretch of sequence has been shown to occur as an extended loop, part of which is involved in inter-subunit interactions; (iii) the amino acid sequence in the region of a second S. cerevisiae inter-subunit contact (residues 74-78) shows radical mutations in the S. pombe enzyme.

Effects of hyperprolactinemia on plasma prolactin and glucose and on local cerebral glucose utilization.

Elevated blood levels of prolactin increase the synthesis, turnover, and release of 3,4-dihydroxyphenylethylamine (dopamine) from the tuberoinfundibular dopaminergic neurons, which project to the median eminence. The present study examined whether hyperprolactinemia also increases local cerebral glucose utilization, as determined by the 2-deoxy-D-[1-14C]glucose method, in the median eminence and other brain structures. Adult male rats were given ovine prolactin (4 mg/kg) subcutaneously every 8 h for 48 h. This treatment exerted an autoregulatory feedback effect on endogenous rat prolactin secretion, as evidenced by decreased circulating levels of rat prolactin. Ovine prolactin treatment also decreased plasma glucose concentrations. However, in both partially immobilized and free-ranging rats, glucose utilization in brain structures containing tuberoinfundibular dopaminergic cell bodies (the arcuate nucleus) and terminals (the median eminence) was not affected by ovine prolactin treatment. Hyperprolactinemia was, however, associated with decreased glucose utilization in the medial forebrain bundle and the CA subfield of the dorsal hippocampus. The lack of a significant effect of prolactin treatment on glucose utilization in the median eminence indicates that the resolution of the deoxyglucose technique, as used here, is not adequate to detect the ovine prolactin-induced increase in tuberoinfundibular dopaminergic neuronal activity, that the median eminence does not utilize glucose as its primary energy substrate, or that ovine prolactin treatment causes a counterbalancing decrease in the activity of other neurons projecting to the median eminence.

Studies on the effects of intrathalamically injected DADL and morphine on nociceptive thresholds and electroencephalographic activity: a thalamic delta receptor syndrome.

Bilateral microinjections of DADL (D-Ala2-D-Leu5-enkephalin) and morphine were carried out in rats in a systematic fashion at histologically identified medial and lateral thalamic sites. DADL produced a dose-dependent (1.5-15.0 nmol), naloxone-reversible (1 mg/kg, i.p.) increase in the hot-plate (HP), tail-flick (TF) and catalepsy (CAT) response latencies with a predominance of activity occurring at lateral as opposed to medial thalamic sites. These effects were seen within 5 min of microinjection. At a significant number of sites, DADL precipitated convulsive seizure activity. Equimolar doses of morphine had a negligible effect on nociceptive indices and were not productive of seizures even at sites where DADL was found to be active. To further examine seizure activity, rats were prepared with bilateral frontal cortical electrodes and microinjected also at medial and lateral thalamic sites with equimolar doses of DADL and morphine (15 nmol). DADL was found to produce electrographically defined seizures unaccompanied by convulsive motor behavior (cataleptic seizures), as well as convulsive seizures. All animals in this group exhibiting analgesia and catalepsy had electrographic evidence of a seizure with markedly abnormal EEG tracings showing postictal spiking and changes in baseline frequency and amplitude. These seizures appeared to be naloxone-reversible. Morphine on the other hand was not productive of seizures, but did produce changes in electroencephalographic activity including spindle bursting, high-voltage slow-frequency activity as well as spiking. As noted, these changes were not associated with any effects on nociceptive measures.