D. B. Ramsden

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Human cysteine dioxygenase type I: primary structure derived from base sequencing of cDNA

The base sequence of cDNA encoding the complete human liver cysteine dioxygenase type I (CDO-I; EC 1.13.11.20) message, and the derived amino-acid sequence are reported. CDO-I is encoded on a single mRNA species (approx. 1.5 kb). Human CDO-I clones were identified by screening a liver cDNA library, and inserts were isolated and sequenced. In addition, human liver total RNA was reverse-transcribed and CDO-I cDNA amplified by PCR using a modified poly-T primer and specific CDO-I primers to give a second source of sequencing template. The CDO-I message encodes a 200 amino-acid residue protein, the sequence of which has greater than 90% homology with the equivalent rat enzyme. The message was not expressed in a human hepatoma cell line (Hep G2).

Measurement of free thyroid hormones in patients on long-term phenytoin therapy

SummaryTotal thyroxine and triiodothyronine levels are often reduced in long-term phenytoin therapy, a finding at variance with the euthyroid clinical state of these subjects. We have measured free (biologically active) thyroid hormone levels in 31 patients on phenytoin therapy and have found a significant reduction in measurements of both free thyroxine and free triiodothyronine. In contrast, addition of phenytoin to normal serum in vitro resulted in an increase in free thyroxine and free triiodothyronine concentrations. The findings in vitro are consistent with displacement of thyroid hormones from thyroxine binding globulin while the reduced free hormone levels in vivo confirm that the effects of phenytoin are not confined to binding inhibition and suggest that phenytoin has induced a change in the cellular metabolism of thyroid hormones.

Serum tetraiodothyroacetate (T4A) levels in normal healthy euthyroid individuals determined by gas chromatography-mass fragmentography (GC-MF)

A gas chromatography-mass fragmentography (GC-MF) assay for T4A in human serum is described. The assay involves a multistage extraction, followed by conversion of T4A to its dimethyl derivative prior to GC-MF. Using this assay, the concentration of T4A in 37 normal euthyroid subjects was, mean +/- S.D., 114.5 +/- 26.4 pmol/l, which is considerably lower than previous reports. Thyroxine binding prealbumin (TBPA) did not appear to be a major determinant of T4A concentration.

THYROXINE‐BINDING PREALBUMIN GENE POLYMORPHISM: A POPULATION STUDY

The human thyroxine‐binding prealbumin (TBPA) gene was examined for restriction fragment length polymorphism (RFLP) in normal subjects and a subject with euthyroid hyperthyroxinaemia, due to increased thyroxine binding by TBPA, using 16 restriction enzymes. Only Taq I and Msp I were shown to detect RFLPs. In a male of the normal population and one of his daughters, an additional Taq I site was found in the 3|M′‐flanking region of the TBPA gene. The RFLP in a subject with euthyroid hyperthyroxinaemia was due to the deletion of a MspI site. All three subjects with RFLPs were heterozygous.

IS OLEIC ACID THE THYROXINE BINDING INHIBITOR IN THE SERUM OF ILL PATIENTS

The possibility that oleic acid is the thyroxine binding inhibitor in the serum of seriously ill patients was investigated. 3H‐Oleic acid was shown to bind directly to human thyroxine‐binding globulin (TBG) by the techniques of one and two‐dimensional immunoelectrophoresis in combination with autoradiography. However, no correlation was seen between serum thyroxine concentration and oleic acid concentration in two groups of patients, one of which underwent routine cholecystectomy, whilst the other group was admitted to an intensive therapy unit (mortality 75%). No correlation was seen between serum total thyroxine concentration and either stearic, palmitic, linoleic or arachidonic acid concentrations in these groups. Therefore, it was concluded that oleic acid was unlikely to be the circulating inhibitor of thyroxine binding.

3,5-Diiodotyrosine and thyronine in the urine of patients with chronic renal disease.

Urinary 3,5‐diiodotyrosine (DIT) and thyronine (T0) excretion was investigated in 18 patients with chronic renal disease. In accord with previous findings serum T4 and thyroid hormone binding proteins measured in 17 patients were in the low or normal range. Urinary albumin excretion was elevated in all 18 and T4 binding prealbumin (TBPA) in 15 of the 18. Urinary T0 excretion measured in 12 patients was also significantly lower than normal (mean±SD 4.4±2.6 vs 15.8±5.8 nmol/24 h renal vs normal 2 P<0.001). In contrast urinary DIT excretion was significantly elevated in renal patients compared with normal subjects (2.0±1.5 vs 0.75±0.41 nmol/24 h, respectively). Possible sources of the increased DIT are discussed.

Glycosylation of human thyroglobulin and characterization by lectin affinity electrophoresis

Thyroglobulin, a 660 kDa glycoprotein, is the major product of protein synthesis in the thyroid gland. It has been suggested that modifications of thyroglobulin glycosylation occur in various thyroid disorders. In order to study possible changes in glycosylation of tissue thyroglobulin associated with thyroid disease, we have developed a lectin affinity electrophoresis system which allows characterization of small (less than 1 microgram) quantities of thyroglobulin. Human thyroglobulin was extracted and purified. Agarose gels were cast containing concanavalin A, Ricinus communis agglutinin, L-phytohaemagglutinin and pokeweed mitogen at various concentrations. Purified human thyroglobulin was serially diluted, loaded onto lectin gels and electrophoresed. Concanavalin A, R. communis agglutinin and phytohaemagglutinin all bound thyroglobulin in a concentration-dependent manner. Pokeweed mitogen did not bind thyroglobulin. Purified thyroglobulin was treated with neuraminidase and endoglycosidase H. Two-dimensional immunoelectrophoresis revealed the migration of thyroglobulin to be modified by neuraminidase but not by endoglycosidase H. Lectin affinity electrophoresis of purified human thyroglobulin with and without enzyme treatment indicated the presence of: oligomannose structures as shown by concanavalin A reactivity and modification by endoglycosidase H, and complex oligosaccharides as shown by affinity for R. communis agglutinin and modification by neuraminidase. These structures are in keeping with the proposed patterns of glycosylation of human thyroglobulin and indicate suitability of the method for characterizing the glycosylation of small quantities of thyroglobulin.

MEASUREMENT OF FREE THYROXINE AND FREE TRIIODOTHYRONINE IN ASYMPTOMATIC SUBJECTS WITH HIGH CONCENTRATIONS OF SERUM TSH AND A HISTORY OF RADIOIODINE THERAPY

Total and free thyroid hormones were measured in a group of asymptomatic subjects with high circulating TSH concentrations and a previous history of radioiodine therapy. Subjects in the high TSH group all fell within the normal range for measurements of total T4, total T3 and T4/TBG ratio. Concentrations of free T4 or free T3 were below the normal range in more than half (9 out of 17) of the high TSH group suggesting that these measurements are more sensitive indices of thyroid failure than measurement of total hormones or T4/TBG ratio.

The assay of thyroxine binding globulin in human serum using nephelometry

A nephelometric assay for the determination of thyroxine binding globulin concentration in human serum is described. This has similar sensitivity to other non-radioactive protein assay, is rapid and highly reproducible. By use of a rate of reaction rather than end point technique the problem of high background interference is circumvented.