An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes

Context: Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia ( typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A.(1) -> G.(1) substitution in intron 6), resulting in the activation of a pseudoexon (6 Psi) and inclusion of 36 amino acids. Objective: The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6 Psi) mutation. Design/Patients: Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed. Main Outcome Measures: We assessed genotype-phenotype relationship. Results: One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from -3.3 to -6.0 SD and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon. Conclusions: Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

Silyl-Modified Analogues of 2-Tritylpyrrolidine: Synthesis and Applications in Asymmetric Organocatalysis

Silicon-based organocatalysts: In an effort to study the effects of substituting carbon by silicon within the catalyst backbone, we developed an efficient synthesis of (S)-2-triphenylsilylpyrrolidine [(S)-2]. The evaluation of (S)-2 against its carbon analogue (S)-1 in two organocatalytic reactions is complemented by computational studies.

Room temperature synthesis of tri-, tetrasubstituted imidazoles and bis-analogues by mercaptopropylsilica (MPS) in aqueous methanol: application to the synthesis of the drug trifenagrel

The heterogeneous solid catalyst, mercaptopropylsilica (MPS), has been prepared by a modified procedure in water and its structure confirmed by solid state carbon-13 CP-MAS NMR spectrum. This catalyst has been efficiently utilized for the synthesis of a wide variety of tri-, tetrasubstituted imidazoles and their bis-analogues at room temperature. The protocol was further explored for the synthesis of the drug trifenagrel.

Differences in glucose-dependent insulinotropic polypeptide hormone and hepatic lipase in subjects of southern and northern Europe: implications for postprandial lipaemia

The aim was to determine in 32 healthy young men from northern and southern Europe whether differences in the secretion of insulin and glucose-dependent insulinotropic polypeptide (GIP) might explain these findings through the actions of these hormones on lipoprotein lipase. In a randomized, single-blind, crossover study the effects of 2 test meals of identical macronutrient composition but different saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents were investigated on postprandial GIP, insulin, the ratio of incremental triacylglycerol to apolipoprotein B-48 (a marker of chylomicron size), and the activity of postheparin lipases. Fasting and postprandial GIP concentrations and postheparin hepatic lipase (HL) activities were higher in the southern Europeans (P<0.001 and P<0.02, respectively). Lipoprotein lipase activity after the SFA-rich meal was higher in the northern Europeans (P<0.01). HL activity 9 h after the SFA-rich meal and the area under the curve (AUC) for the postprandial insulin response correlated with the AUC for the postprandial GIP response (r=0.44 (P<0.04) and r=0.46 (P<0.05), respectively). There were no significant differences in chylomicron size between the 2 groups for either meal, but when the groups were combined there was a difference in chylomicron size between the SFA- and MUFA-rich meals (P<0.05), which could be due to the formation of larger chylomicrons after the MUFA-rich meal. The significantly higher GIP and insulin responses and HL activities in southern Europeans may provide an explanation for a previous report of attenuated postprandial triacylglycerol and apolipoprotein B-48 responses in them.

Postprandial lipid and hormone responses to meals of varying fat contents: modulatory role of lipoprotein lipase?

OBJECTIVE: Substrate and hormone responses to meals of differing fat content were evaluated in normal subjects in order to investigate mechanisms underlying the regulation of postprandial lipoprotein concentration. DESIGN: A randomised cross-over study with three different meals on three occasions. SETTING: Free-living subjects associated with Surrey University. SUBJECTS: Ten male volunteers (aged 18-23 years) were recruited. INTERVENTIONS: Three test meals containing 20, 40 or 80 g fat but identical carbohydrate and protein content were randomly allocated to volunteers. MAJOR OUTCOME MEASURES: Pre- and postprandial blood samples were taken for the analysis of plasma triacylglycerol, non-esterified fatty acids, glucose, immunoreactive insulin and glucose-dependent insulinotrophic polypeptide levels and postheparin lipoprotein lipase activity measurements. RESULTS: Peak triacylglycerol concentrations and lipoprotein lipase activity measurements were significantly higher following the 80 g than the 20 g fat meal (P = 0.009 and P = 0.049 respectively). Areas under the glucose-dependent insulinotrophic polypeptide time-response concentration curves were significantly higher following the 80 g compared with the 20 g fat meal (P = 0.04), but no differences in insulin response to the meals were seen. The 30-360 min decrease in the non-esterified fatty acid concentration was less following the 80 g than the 20 g meal (P = 0.001). CONCLUSIONS: The results suggest that glucose-dependent insulinotrophic polypeptide may mediate increased lipoprotein lipase activity in response to fat-containing meals and may play a role in circulating lipoprotein homeostasis. This mechanism may be overloaded with high fat meals with adverse consequences on circulating triacylglycerol and NEFA concentrations.

The effect of triacylglycerol fatty acid positional distribution on postprandial plasma metabolite and hormone responses in normal adult men

The present study has examined the possibility that the positional distribution of fatty acids on dietary triacyglycerol (TAG) influences the postprandial response to a liquid meal in adult subjects. Postprandial TAG, non-esterified fatty acids (NEFA), ketones, glucose, insulin and gastric inhibitory polypeptide (GIP) responses were monitored in sixteen normal adult male subjects over 6 h following consumption of test meals containing dietary TAG in which palmitic acid was predominantly on the sn-1 (Control) or sn-2 positions (Betapol). Plasma total TAG, chylomicron-rich TAG and chylomicron-poor TAG concentrations were identical in response to the two test meals. The peak increase (mean (SD)) in chylomicron TAG was 0.85 (0.46) mmol/l after the Control meal and 0.85 (0.42) mmol/l after the Betapol meal. Plasma glucose, insulin, GIP, NEFA and ketone concentrations were also very similar following the two meals. It is concluded that dietary TAG containing saturated fatty acids on the sn-2 position appear in plasma at a similar level and over a similar timescale to TAG in which saturated fatty acids are predominantly located on sn-1 or sn-3 positions. The results reported in the present study demonstrate that the positional distribution of fatty acids on dietary TAG is not an important determinant of postprandial lipaemia in adult male subjects, but do not exclude the possibility that different responses may occur when these dietary TAG are given long term.

Effects of n-3 fatty acids on postprandial triacylglycerol and hormone concentrations in normal subjects

The present study reports results from two investigations to determine effects of a 6-week period of moderate n-3 fatty acid supplementation (2.7 g/d) on fasting and on postprandial triacylglycerol and metabolic hormone concentrations in response to standard test meals. In the first study postprandial responses were followed for 210 min after an early morning test meal challenge; in the second study responses to an evening test meal were followed during the evening and overnight for a total period of 12 h. In both studies postprandial triacylglycerol responses to the test meals were significantly reduced after compared with before fish-oil supplementation. In the second study the triacylglycerol peak response seen between 200 and 400 min in subjects studied before supplementation with fish oils was almost completely absent in the same subjects after 6 weeks of n-3 fatty acid supplementation. Analysis of fasting concentrations of metabolites and hormones was carried out on the combined data from the two studies. There were no significant differences in total, low-density-lipoprotein- or high-density-lipoprotein-cholesterol concentrations during fish-oil supplementation, although there was considerable individual variation in cholesterol responses to the supplement. Concentrations of Apo-B and Apo-A1 were unchanged during supplementation with fish oils. Fasting and early morning postprandial GIP concentrations were lower in subjects taking fish oils, possibly due to acute effects of fish-oil capsules taken on the evening before the studies. In both studies fasting insulin and glucose and postprandial insulin concentrations remained unchanged following fish-oil supplementation. The results do not support the view that triacylglycerol-lowering effects of n-3 fatty acids are due to modulation of insulin secretion mediated via the enteroinsular axis. Further studies are required to determine the precise mechanism by which fish oils reduce both fasting and postprandial triacylglycerol concentrations.

Thermogenic and metabolic consequences of thyroid hormone treatment in brown and white adipose tissue

Male rats were treated with triiodothyronine in the drinking water for 12 days. In vitro rates of isoprenaline stimulated lipolysis were significantly greater in brown but not white adipose tissue. Rates of [14C]glucose incorporation into triacylglycerols were significantly reduced in BAT (brown adipose tissue) and WAT (white adipose tissue) under basal and isoprenaline stimulated conditions. In a second experiment, hyperthyroid animals showed impaired weight gain, despite increased food intake during 19 days' treatment. Energy expenditure on days 5 and 12, and BAT core temperature differences (TBAT - TCORE) on day 19, were significantly greater than in control animals. Epididymal white fat pad weight was reduced and interscapular brown fat pad weight increased by triiodothyronine treatment.

Approaches to the synthesis of novel photoactive side chain liquid crystalline polymers containing analogues of the cinnamate ester group

A series of chain liquid crystalline copolymers of 4-cyanophenyl 4′-(6-methacryloyloxyhexyloxy)benzoate and 2-methacryloyloxyethyl β-(1-naphthyl)-propenoate were prepared by free radical polymerization. The corresponding polyacrylates could not be prepared in the same way and an alternative method was used for their preparation involving the synthesis of copolymers of the mesogenic monomer and 2-hydroxyethyl acrylate followed by treatment of the resulting polymers with β-(1-naphthyl)propenoyl chloride. The materials are of interest as photoactive liquid crystalline polymers. The effect of introducing a bulky nonmesogenic group into a liquid crystalline copolymer generally lowers the clearing temperature and raises Tg but also gives rise to contrasting phase behaviour in these two series of polymers. Polymethacrylates which show mesomorphism have sharp transitions and continue to exhibit a highly ordered smectic phase over the bulk of their liquid crystal range. Polyacrylates, on the other hand, exhibit a weakening and broadening-out of their thermal transitions consistent with a lowering of order. These results emphasize the effect of the polymer backbone on phase behaviour.

Strigolactone analogues induce suicidal seed germination of Striga spp. in soil

Striga hermonthica and Striga asiatica are obligate root parasites that cause serious problems in the production of staple cereal crops in Africa. Because of the high levels of infestation, there is an urgent need to control these weeds. A potentially useful control option is depletion of the soil seed bank by suicidal germination, which involves germination of the seeds in the absence of host plants. Suicidal germination is often mentioned in the literature, but not considered realistic, because of the alleged untimely decomposition of the stimulants in the soil, despite the fact that some encouraging results were reported around 1980. The alleged instability has prevented active research in this direction for the past 20–25 years. Five newly designed synthetic germination stimulants were investigated as candidates for suicidal germination. An important issue is the persistence of these stimulants in soil. Packets with Striga spp. seeds were put in pots with soil and then treated with aqueous solutions of the stimulants. All five compounds induced germination under these conditions, with percentages varying between 18% and 98% depending on stimulant and species. There were no noticeable signs of decomposition of the stimulants. The best performing stimulant is derived from 1-tetralone. We conclude that synthetic strigolactones analogues have excellent prospects for use in combating parasitic weeds. Further testing will be needed to evaluate whether such prospects can be realised in the field.

Developing skin analogues for a robotic octopus

In order to fabricate a biomimetic skin for an octopus inspired robot, a new process was developed based on mechanical properties measured from real octopus skin. Various knitted nylon textiles were tested and the one of 10-denier nylon was chosen as reinforcement. A combination of Ecoflex 0030 and 0010 silicone rubbers was used as matrix of the composite to obtain the right stiffness for the skin-analogue system. The open mould fabrication process developed allows air bubble to escape easily and the artificial skin produced was thin and waterproof. Material properties of the biomimetic skin were characterised using static tensile and instrumented scissors cutting tests. The Young’s moduli of the artificial skin are 0.08 MPa and 0.13 MPa in the longitudinal and transverse directions, which are much lower than those of the octopus skin. The strength and fracture toughness of the artificial skin, on the other hand are higher than those of real octopus skins. Conically-shaped skin prototypes to be used to cover the robotic arm unit were manufactured and tested. The biomimetic skin prototype was stiff enough to maintain it conical shape when filled with water. The driving force for elongation was reduced significantly compared with previous prototypes.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.