Expression of SHOX in human fetal and childhood growth plate.

Abnormalities in the growth plate may lead to short stature and skeletal deformity including Leri Weil syndrome, which has been shown to result from deletions or mutations in the SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome. We studied the expression of SHOX protein, by immunohistochemistry, in human fetal and childhood growth plates and mRNA by in situ hybridization in childhood normal and Leri Weil growth plate. SHOX protein was found in reserve, proliferative, and hypertrophic zones of fetal growth plate from 12 wk to term and childhood control and Leri Weil growth plates. The pattern of immunostaining in the proliferative zone of childhood growth plate was patchy, with more intense uniform immunostaining in the hypertrophic zone. In situ hybridization studies of childhood growth plate demonstrated SHOX mRNA expression throughout the growth plate. No difference in the pattern of SHOX protein or mRNA expression was seen between the control and Leri Weil growth plate. These findings suggest that SHOX plays a role in chondrocyte function in the growth plate.

Ocular myasthenia gravis: predictive value of single-fiber electromyography.

Extraocular muscle weakness is the most common presenting sign of myasthenia gravis (MG). More than half of patients presenting with symptoms isolated to these muscles (OMG) develop generalized myasthenia gravis (GMG) over the course of their illness. No clinical, laboratory, or electrophysiological features are recognized that identify these high-risk patients. We have therefore assessed the ability of single-fiber electromyography (SFEMG) to predict the development of GMG in patients presenting with OMG. Thirty-nine consecutive patients presenting with OMG underwent SFEMG of the extensor digitorum communis muscle as well as a battery of other laboratory and imaging studies at the time of diagnosis. All patients were followed prospectively for a minimum of 24 months or until they developed GMG. Two patients were excluded, leaving 37 for assessment. Twenty remained with pure OMG for the entire follow-up period (mean, 55 months). Twenty-six of the 37 had abnormal SFEMG studies at presentation. Eleven of these remained with OMG and 15 developed GMG. Fifty-eight percent of patients with an abnormal SFEMG developed GMG, whereas 82% of those with a normal study remained with OMG. Thus, a normal SFEMG was associated with MG remaining restricted to the extraocular muscles. (P = 0.036, Fisher's exact test), but an abnormal SFEMG was not predictive of subsequent development of GMG.

In vitro evaluation of fotemustine as a potential agent for limb perfusion in melanoma.

The mechanism of action of fotemustine, a relatively new chloroethylnitrosourea, was evaluated in human melanoma cells in order to assess its potential as an agent for hyperthermic limb perfusion. Fotemustine was more toxic to O6-alkylguanine methyl transferase (AGT) deficient (Mer-) cells than Mer+ cells, implicating AGT as a major determinant of resistance. Mer+ cells derived from Mer- cell lines following exposure to the monofunctional alkylating metabolite of dacarbazine (DTIC) were also resistant to fotemustine. Mer status did not influence the replication of fotemustine-damaged adenovirus 5, whereas virus treated with the monofunctional alkylating agent N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) was replicated much more efficiently by Mer+ cells. This suggests that the initial O6-alkylated product, if not immediately repaired, rearranges to form DNA crosslinks which cannot be repaired by AGT. Replication of a control virus was not affected by treating the cells with fotemustine, indicating that the drug acted primarily on DNA rather than at epigenetic levels. Fotemustine generally produced a G2-M block in the cell cycle, most strikingly in Mer- cells at low, minimally toxic concentrations; MNNG and high doses of fotemustine induced S phase arrest. Concurrent hyperthermia (41.5 degrees C for 1 h) increased the toxicity of fotemustine in some cell lines. Fotemustine decomposed in culture medium in two phases; the first was complete within 5 min and was most marked in Mer+ cells. The results suggest that fotemustine may be suitable for isolated limb perfusion in melanoma, with the potential for overcoming resistance by including inhibitors of AGT.

Mechanism of action of fotemustine, a new chloroethylnitrosourea anticancer agent: evidence for the formation of two DNA-reactive intermediates contributing to cytotoxicity.

Methyl excision repair deficient human tumor cells (Mer-) were found to be hypersusceptible to killing by the antimelanoma agent fotemustine (FM) implicating alkylation of O6 guanine as the major contributor to toxicity. Preincubation of the drug in aqueous solution for 5 min resulted in an immediate reduction in cytotoxicity (35-50%), in vitro DNA alkylation (31%), and DNA interstrand cross-linking (40%) followed by a second reaction with considerably slower kinetics. Electrospray ionisation mass spectrometry (ESI-MS) showed that in aqueous solution FM rearranged rapidly to form either a metastable tautomer or decomposed to form a highly reactive diazohydroxide (t1/2 < 2 min). These results suggest the presence of two DNA-reactive species relevant to biological activity. Coincubation of ellagic acid (an inhibitor of O6-guanine alkylation) with FM inhibited in vitro ISC, suggesting that the O6-chloroethyl lesion is the predominant cause of the cross-link. On the basis of these findings, we propose that FM breaks down to form a short-lived intermediate, 2-chloroethyldiazohydroxide, which rapidly generates O6-guanine lesions responsible for the drug's initial activity and a long lived iminol tautomer responsible for the remaining O6 guanine alkylation and cytotoxicity.

NMR study of the interaction of plastocyanin with chromium(II) analogues of inorganic electron transfer reagents.

High resolution nuclear magnetic resonance spectroscopy has been used to examine the interaction of plastocyanins from French bean (Phaseolus vulgaris) and cucumber (Cucumis sativus) with three complexes--potassium hexacyano-chromate(III), hexamminechromium(III) nitrate and tris(1,10-phenanthroline)-chromium(III) perchlorate--which are analogues of inorganic electron transfer reagents. The results indicate a high degree of specificity in the binding of these complexes and two binding sites on the protein are identified. One binding site is situated close to the copper atom and is clearly suited to outer sphere electron transfer through one of the histidine ligands. The other binding site is more distant from the copper atom and this mechanism cannot be operative. Electron transfer via hydrophobic channels or electron tunneling are possible mechanisms of electron transfer.