Recombinant human thyroid-stimulating hormone (Thyrogen) in thyroid cancer follow up: experience at a single institution.

BACKGROUND: Recombinant human thyroid-stimulating hormone (Thyrogen; Genzyme Corporation, Cambridge, MA, USA) (rhTSH)-stimulated serum thyroglobulin (Tg) (stim-Tg) and (131)I whole-body scanning (WBS) have been reported to allow follow up of patients with thyroid cancer without the symptoms of thyroxine withdrawal and with equivalent diagnostic information to that obtained after thyroxine withdrawal. The aim of the study was to report results of rhTSH use at the Alfred Hospital, Melbourne, from 1999 to 2006 and in particular to examine the significance of detectable serum Tg after rhTSH in relation to thyroid cancer staging and to compare the sensitivity of rhTSH-stimulated serum Tg to whole-body (131)I scanning (WBS) in the detection of residual and recurrent thyroid cancer. METHODS: The study was a retrospective chart review. RESULTS: In 90 patients, rhTSH was used for 96 diagnostic episodes and 18 doses of rhTSH were used to facilitate treatment with (131)I. In stages I and II cancer (n = 42), of three patients with stim-Tg 1-2 microg/L, none had identifiable disease, and the three patients who had stim-Tg >2 microg/L did not experience recurrent disease during follow up. In contrast, in stages III and IV cancer (n = 43) 2 of 5 with stim-Tg 1-2 microg/L had identifiable disease and 7 of 10 with stim-Tg >2 microg/L had identifiable disease. In Tg-positive, WBS-negative disease, further imaging identified persistent/recurrent disease. CONCLUSION: rhTSH was effective and safe in the management of thyroid cancer follow up for diagnosis of persistent/recurrent cancer and to enable (131)I treatment. In no case did rhTSH-stimulated WBS identify the presence of disease not also identified by raised basal Tg or stim-Tg. Therefore, in low risk cancer WBS may be omitted.

Heterogeneity of amiodarone-induced thyrotoxicosis: evaluation of colour-flow Doppler sonography in predicting therapeutic response.

BACKGROUND: Amiodarone-induced thyrotoxicosis (AIT) presents a therapeutic challenge because of its resistance to standard antithyroid therapy. In iodine-deplete environments, colour-flow Doppler sonography (CFDS) has allowed distinction between two types of AIT: (i) Type I AIT, associated with increased vascularity (CFDS I-III) and response to thionamide antithyroid drug and (ii) type II AIT, with no/little thyroid vascularity (CFDS 0) and prednisolone responsiveness. AIM: To clarify if CFDS patterns correlated with treatment outcomes in a retrospective study of 24 patients with AIT in an iodine-replete environment. METHODS: Medical records of patients who presented to a teaching hospital between January 1998 to December 2000 were reviewed. Results of CFDS, ultrasound measurement of thyroid size and technetium scanning of the thyroid were correlated with treatment responses, especially prednisolone responsiveness. RESULTS: Thirteen of 24 patients showed CFDS 0. Twelve of these 13 were evaluable for prednisolone responsiveness, of whom seven (58%) were prednisolone-responsive. Of 11 patients with CFDS I-III, four (36%) responded to antithyroid medication alone and only one of seven (14%) was prednisolone-responsive. Euthyroidism was achieved twice as rapidly in patients with CFDS 0 than those with CFDS I-III. Because of medical treatment failure, seven patients, from both CFDS groups, required urgent near-total thyroidectomy which was successful and uncomplicated in all cases. CONCLUSIONS: CFDS is useful in the management of AIT because CFDS 0 correlates better with prednisolone response (58%) than CFDS I-III (14%). However, unlike experience in iodine-deficient regions, the results of the present study revealed that treatment responses to thionamide or prednisolone were heterogeneous within uniform CFDS patterns. Thus, prednisolone--responsiveness was not consistently predicted by CFDS 0, but the presence of flow appeared to correlate with non-response to prednisolone.

Development of an efficient system for the separation of indole alkaloids by high performance liquid chromatography and its applications.

An efficient system for the analysis of indole alkaloids by HPLC on a reversed-phase column using an ion pair technique is described. The optimised chromatographic conditions allowed the successful separation of 22 standard monoterpenoid indole alkaloids (including some isomers) and tryptamine. The described HPLC system was applied to the analysis of alkaloids in intergeneric somatic hybrid cell cultures of Rauvolfia serpentina x Rhazya stricta.

Glucosylation of isatin-3-oxime followed by 2D in situ NMR in plant cells at highest magnetic field without labelling.

The glucosylation of isatin-3-oxime (1) was monitored by in situ 2D 1H-13C inverse correlated gradient assisted NMR spectroscopy in plant cell suspension cultures of Rauvolfia serpentina without labelling. The applied high magnetic field of 800 MHz allowed measurements within 20 min at concentrations of 1 of 5.76 mM. Complete glucosylation of 1 occurs inside the cells within 72 hours. During this time isatin-3-oxime-glucoside (2) accumulates without further metabolism.

Utilizing genetically engineered bacteria to produce plant-specific glucosides.

Plant-derived glucosides have attracted much attention due to their widespread applications. This class of products is difficult to isolate or to synthesize in pure form because of the resulting low yields. Thus, simple approaches for the generation of such glucosides would be highly beneficial. We purified and characterized a novel glucosyltransferase from plant cell suspension cultures of Rauvolfia serpentina, which showed rather low substrate specificity. We obtained its cDNA and expressed the active recombinant protein in bacteria (Escherichia coli) with excellent plant-specific glucosylation efficiencies. Compared with the plant system, the bacteria delivered the new enzyme, which was in the form of a soluble or matrix-bound enzyme, approximately 1800 times more efficiently for the synthesis of a wide range of glucosides. More importantly, the engineered E. coli strain allowed for in vivo glucosylation and release of the product into the culture medium, as shown by the formation of arbutin, which is a potent inhibitor of human melanin biosynthesis with commercial value.

Free thyroid hormone measurement. A critical appraisal.

The main purpose of free T4 and free T3 assays is to distinguish reliably between thyrotoxicosis, hypothyroidism, and the euthyroid state, an objective that cannot be attained with assays of total T4 and T3 because of hereditary and acquired variations in the concentrations of binding proteins. Effective correction for changes in the serum concentration of TBG can be achieved with numerous types of free hormone estimate, but other changes in binding are not well accommodated. Despite remarkable methodologic ingenuity, no current method reflects the free T4 concentration in undiluted serum under in vivo conditions. Equilibrium dialysis, widely considered the reference method for free T4 measurement, is also subject to error, either preanalytic, owing to generation of NEFA in the sample leading to an overestimate of free T4, or analytic with underestimation of the effect of competitors to increase free T4. Current approaches to free T4 measurement are vulnerable to several method-dependent artifacts: abnormal albumin binding of T4 or of the assay tracer, the inhibition of T4 binding to TBG by medications, and the effects of critical illness, especially in heparin-treated patients, pregnancy, and the abnormalities in sick premature infants. Because of systematic variation between methods (i.e., whether a technique is albumin dependent or prone to incubation or dilution artifacts), it is essential to consider methodologic details in evaluating free T4 estimates in these situations and whenever estimates of free T4 are clinically discordant. False-positive abnormalities are more frequent than false-negative results. When free T4 results are correlated with the serum TSH concentration with attention to the assumptions that define this relationship, the majority of false-positive results can be readily identified. If a free T4 anomaly remains unexplained on repeat sampling, it is appropriate to use an alternative free T4 method that depends on a different assay principle and to correlate the result with an authentic total T4 measurement.

High yielding one-pot enzyme-catalyzed synthesis of UDP-glucose in gram scales.

Uridine diphosphoglucose is an important cofactor of glucosylating enzymes. A simple and high yielding one-pot enzymatic synthesis of UDPG on a gram scale from glucose via hexokinase, phosphoglucomutase and UDPG pyrophosphorylase (UGPase) is described. Repetitive addition of substrate was used to avoid inhibition of UGPase. The approach allows recovery of active enzymes and their re-use. The synthesis of UDP-[4-(13)C]-glucose on a 0.5 g scale resulted in a final yield of 70% and a purity of >95% after chromatographic purification.

3-oxo-rhazinilam: a new indole alkaloid from Rauvolfia serpentina x Rhazya stricta hybrid plant cell cultures.

A new monoterpenoid indole alkaloid, 3-oxo-rhazinilam (1), was isolated from intergeneric somatic hybrid cell cultures of Rauvolfia serpentina and Rhazya stricta, and the structure was determined by detailed 1D and 2D NMR analysis. It was also proved that 3-oxo-rhazinilam (1) is a natural constituent of the hybrid cells.

Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis.

Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.

The structure of the ring-opened N beta-propyl-ajmaline (Neo-Gilurytmal) at physiological pH is obviously responsible for its better absorption and bioavailability when compared with ajmaline (Gilurytmal).

Prajmaline, the semisynthetic propyl derivative of ajmaline, shows a much better bioavailability when compared with the Rauvolfia alkaloid ajmaline. Early NMR and IR-studies, fluorescence spectroscopic investigations and extraction experiments combined with ion-pair chromatography proved the thesis of a tautomeric equilibrium between an aldehyde-amine and a quaternary carbinol-ammonium component. The aim of this study was to confirm this thesis by HPLC-separation and by structure-determination of both tautomeric compounds.

Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences.

Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone. A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase. SDS-PAGE showed a M(r) for the enzyme of 52 kDa. Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced. Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.

The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha/betahydrolase super family.

The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.

Tooth root resorption associated with a familial bone dysplasia affecting mother and daughter.

The dental findings are presented of a mother and daughter who suffer from an as yet unclassified bone dysplasia that shows features of both hereditary hyperphosphatasia and familial expansile osteolysis. Both patients have experienced progressive root resorption of permanent teeth, deafness, and high alkaline phosphatase levels. The mother has a more advanced bone dysplasia which has led to progressive skeletal deformity and bone pain. The kindred is consistent with an autosomal dominant pattern, and the mutation(s) is thought to be in chromosome 18q21-22 region. Conventional treatment strategies of root resorption offer only a poor prognosis for the dentition. Therapy using alendronate, a bisphosphonate compound and a potent inhibitor of osteoclastic activity, has reduced alkaline phosphatase levels, bone pain, and may offer an effective strategy to prevent tooth root resorption in this group of diseases.

Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina.

Plant cell suspension cultures of Rauwolfia produce within 1 week approximately 250 nkat/l of raucaffricine-O-beta-D-glucosidase. A five step procedure using anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yield of 0.9% approximately 1200-fold enriched glucosidase. A short protocol employing DEAE sepharose, TSK 55 S gel chromatography and purification on Mono Q gave a 5% recovery of glucosidase which was 340-fold enriched. SDS-PAGE showed a Mr for the enzyme of 61 kDa. The enzyme is not glycosylated. Structural investigation of the enzyme product, vomilenine, demonstrated that the alkaloid exists in aqueous solutions in an equilibrium of 21(R)- and 21(S)-vomilenine in a ratio of 3.4:1. Proteolysis of the pure enzyme with endoproteinase Lys C revealed six peptide fragments with 6-24 amino acids which were sequenced. The two largest fragments showed sequences, of which the motif Val-Thr-Glu-Asn-Gly is typical for beta-glucosidases. Sequence alignment of these fragments demonstrated high homologies to linamarase from Manihot esculenta (81% identity) or to beta-glucosidase from Prunus avium (79% identity). Raucaffricine-O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases.

The Australian Multicenter Trial of Growth Hormone (GH) Treatment in GH-Deficient Adults.

GH treatment in adults with GH deficiency has numerous beneficial effects, but most studies have been small. We report the results of an Australian multicenter, randomized, double-blind, placebo-controlled trial of the effects of recombinant human GH treatment in adults with GH deficiency. GH deficiency was defined as a peak serum GH of < 5 mU/liter in response to insulin-induced hypoglycemia. Patients were randomly assigned to receive either GH (0.125 U/kg per week for 1 month and 0.25 U/kg per week for 5 months) or placebo. After 6 months, all patients received GH. The primary end points were biochemical responses, body composition, quality of life, and safety. One hundred sixty-six patients (72 females and 91 males) with a mean age of 40 +/- 1 yr (+/- SEM; range 17-67 yr) were recruited. Serum insulin-like growth factor-I (IGF-I) increased from a standard deviation score of -2.64 +/- 0.27 (range -8.8 +3.82; n = 78) to +1.08 +/- 2.87 (range -7.21 to +6.42) at 6 months in the GH/GH group; 38% of the whole group were above the age-specific reference range following treatment [17.6% and 68.9% with subnormal (< 2 SD) or normal (+/- 2 SD) pretreatment levels, respectively]. Fasting total cholesterol (P = 0.042) and low-density lipoprotein cholesterol (P = 0.006) decreased over the first 6 months. Fat-free mass increased in the first 6 months whether measured by bioelectrical impedance (P < 0.001) or dual energy x-ray absorptiometry (DEXA; P < 0.001). Total-body water increased in the first 6 months whether measured by bioelectrical impedance (P < 0.001) or deuterium dilution (P = 0.002). Fat mass measured by DEXA (P < 0.001), skinfold thicknesses (P < 0.001), and waist/hip ratio (P = 0.001) decreased in the first 6 months. Most changes in body composition were complete by 3 months of treatment and maintained to 12 months. Whole-body bone mineral density (BMD) (by DEXA) was unaffected by GH treatment. Self-reported quality of life was considered good before treatment, and beneficial treatment effects were observed for energy, pain, and emotional reaction as assessed by the Nottingham Health Profile. In the initial 6 months, adverse effects were reported by 84% of patients in the GH and 75% in the placebo group, with more symptoms relating to fluid retention in the GH group (48% vs. 30%; P = 0.016). Such symptoms were mild and resolved in 70% of patients despite continued treatment. Resting blood pressure did not change over the initial 6 months. In summary, GH treatment in adults with GH deficiency resulted in 1) prominent increases in serum IGF-I at the doses employed, in some cases to supraphysiological levels; 2) modest decreases in total- and low-density lipoprotein cholesterol, together with substantial reductions in total-body and truncal fat mass consistent with an improved cardiovascular risk profile; 3) substantial increases in lean tissue mass; and 4) modest improvements in perceived quality of life. The excessive IGF-I response and side-effect profile suggests that lower doses of GH may be a required for prolonged GH treatment in adults with severe GH deficiency.

Modulation of T3-induced sex hormone-binding globulin secretion by human hepatoblastoma cells.

We have shown previously that tri-iodothyronine (T3)-induced sex hormone-binding globulin (SHBG) secretion by the human hepatoblastoma cell line, HepG2, can be modulated by retinoids. We have now used this model to study a range of other compounds that are known to influence T3 responsiveness in various cell systems. HepG2 cells were incubated for 4 days in serum-free medium containing T3, together with insulin, dexamethasone, phorbol myristate (PMA), sodium butyrate or estradiol. T3 (10 nmol/l) alone induced a concentration of SHBG secreted by HepG2 cells that was 187 +/- 20% (mean +/- S.D., n = 9) of control. Insulin (100 nmol/l) reduced basal SHBG secretion from 24.7 +/- 5.2 nmol/l to 16.1 +/- 1.7 nmol/l (P < 0.01). This effect was dose responsive, half-maximal at 3.4 +/- 3.0 nmol/l (approximately 600 mU/l) and maximal with 100 nmol/l insulin. Co-incubating 0-10 nmol/l T3 with 100 nmol/l insulin resulted in a downward shift in the dose-response curve without a change in the half-maximal response to T3. Conversely, 0-100 nmol/l insulin reduced SHBG production induced by 10 nmol/l T3. In contrast; while dexamethasone alone was without effect on SHBG secretion, 100 nmol/l dexamethasone induced a shift to the left in half-maximal T3 stimulation from 0.37 nmol/l to 0.10 nmol/l. The effect of PMA on SHBG secretion was reminiscent of the previously observed retinoid effect. PMA 100 nmol/l abolished maximal T3 stimulation. This effect was dose responsive, with a threshold at 1 nmol/l PMA. Sodium butyrate, up to 1 mmol/l was without effect; with greater concentrations, SHBG secretion was reduced. T3 responsiveness was virtually abolished by 3 mmol/l sodium butyrate; higher concentrations were cytotoxic and secretion was reduced to less than 20% of basal. Lack of an effect of estradiol on SHBG secretion by HepG2 cells was confirmed. These studies suggest that T3-induced SHBG secretion by HepG2 cells is independently influenced by insulin, potentiated by dexamethasone, and modulated by PMA. Detailed molecular analysis of this model will increase our understanding of the mechanism of action of T3, specifically in human liver cells.