Quantitative determination of biocytin in urine of patients with biotinidase deficiency using high-performance liquid chromatography (HPLC)

A specific method for the quantitative determination of biocytin from urine of biotinidase deficient patients is described using HPLC-separation and quantitative determination by an avidin binding method. Partial purification of biocytin from urine was achieved with an anion exchange resin and concentration of the eluate by lyophilization. The recovery of biocytin from urines was 95.3 +/- 5.9 (mean +/- SD). The precision of biocytin estimation in patients urines including the HPLC-sample preparation procedure varied between 5.9% and 10.5% (CV). Biocytin concentrations were measured in urine samples of 5 patients obtained during and/or before biotin therapy. Before treatment biocytin excretion ranged from 6.2-28.8 nmol/mmol creatinine. During therapy biocytin excretion increased to the 1.3 to 4-fold level in 3 out of 4 patients. However, there was no dose-related increase of biocytin excretion when pharmacological doses were administered. Apart from biocytin and biotin, patients excrete additional biotin derivatives. Some of these have been preliminary identified as bisnorbiotin and oxidation products of bisnorbiotin, biocytin and biotin.     

The Dutch Dental Association's vision of dental training

The Dutch Dental Association (NMT) feels very involved in dental schooling and advocates as broad an education as possible with explicit attention to the 'gatekeeper' function of the dentist in general practice, diagnostics and the development of motor skills for complex, restorative treatments. Attention is demanded to place individual professional practice in a wider context of dental staff and a network of opportunities of referral. The NMT wishes to develop a system for the profession in which guidelines, intercollegial testing and effective accredited provision of further training, systemic opportunities and guidelines for the promotion of expertise are given in the areas of technical methods, organisation of practices and professional attitude. They wish to support the newly-qualified dentist through the introduction of a year of practical training linked to study, so that an 'educational continuum' of education, training and continual further training develops, and to make a contribution not just organizationally, but also regarding the content of the curriculum, certainly where it concerns the sociological aspects and the needs of the professional group.     

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.     

Cancer incidence in a trial of an antiapoptotic agent for Parkinson's disease

We performed a placebo‐controlled trial of CEP‐1347, an inhibitor of neuronal apoptotic cell death, in patients with early Parkinson's disease (PD) to determine whether long‐term therapy would slow disability progression. This also provided an opportunity to monitor cancer incidence in a large cohort of PD patients followed prospectively including periods before and after patients developed disability requiring dopaminergic therapy. This was a multicenter study of 806 patients with early PD, without disability requiring dopaminergic therapy, assigned randomly to placebo or one of three doses of CEP‐1347. Patients were monitored for an average of 1.8 years (1,467 patient‐years) with routine cancer screening evaluations and annual skin examinations by a dermatologist. There was no significant excess of cancers among patients taking CEP‐1347 compared with placebo for any cancer type (all P > 0.1). Nonmelanoma skin cancers were the most common cancer type observed. The incidence of melanomas was 20.9 times that predicted in the general population. Most melanomas occurred in patients who had never taken dopaminergic therapy. We found no evidence that CEP‐1347 affected cancer incidence within 2 years of follow‐up. Melanoma occurrence in our PD patients was greater than predicted compared with the general population and was unrelated to dopaminergic therapy. Clinical surveillance of PD patients for melanoma may be warranted. © 2010 Movement Disorder Society     

Reproductive safety studies with genistein in rats.

Genistein is a phytoestrogen that occurs naturally in the diet and is found in a wide variety of plant-derived foods especially in soybeans and soy-based foods. There is wide spread interest in genistein and related phytoestrogens as chemopreventive agents for a variety of human diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Soy, and hence its constituents, such as genistein, have been consumed at high levels in several Asian populations for many centuries without any apparent adverse effects and to the contrary, many health benefits have been associated with the ingestion of soy based foods. Concern has been raised, however, of potential adverse effects due to the estrogenic and other activities of the isoflavones and thus a comprehensive series of safety studies was performed with genistein. To assess the teratogenic and fetal toxic potential of genistein, several studies were conducted. Genistein was tested in an in vitro rat whole embryo culture assay (WEC), which is a preliminary screen, for fetotoxic and teratogenic potential, over a concentration range of from 1 to 100 microg/mL. Treatment related anomalies were observed at concentrations of >or= 10 microg and at 100 microg/mL, all embryos were malformed. Two in vivo embryo fetal developmental safety studies were conducted with genistein by oral administration (gavage and dietary admix) in which there was no evidence for a teratogenic effect. In an oral (gavage) embryonic and fetal development pilot study, genistein was administered to rats at dose levels of 0, 20, 150 and 1000 mg/kg/day from days 6-20 of gestation to females that were allowed to litter and rear their offspring up to day 7 of lactation. A slight maternal toxicity at 1000 mg/kg/day was observed as indicated by decreased body weight and food consumption and at this dose, adverse effects in the pups were observed including increased pup mortality, poor general condition, reduced pup body weights, and reduced pup milk uptake. At the high dose of 1000 mg/kg, no external malformations were noted, however some minor visceral and skeletal variations were observed. At the low dose of 20 mg/kg/day, an increased mortality, reduced milk uptake, a decreased % male sex ratio, and decreased body weights during lactation were observed. Due to lack of effects at the mid dose and the small number of animals, a relationship to treatment was considered unlikely. In an oral (dietary admix) Prenatal developmental safety study, genistein was administered to rats at dose levels of 0, 5, 50, 100 and 500 mg/kg/day from day 5-21 of gestation. At 500 mg/kg, maternal body weight and food consumption were markedly reduced. The incidence of resorptions was markedly increased with a corresponding decrease in the number of live fetuses per dam. Fetal body weights were also reduced. No treatment-related teratogenic effects were noted during external, visceral and skeletal examination of fetuses or in body weight normalized anogenital distance. On the basis of these studies, it is concluded that genistein has no teratogenic potential in vivo at very high doses of up to 1000 mg/kg/day by oral gavage in the embryo-fetal toxicity pilot study or up to 500 mg/kg/day by dietary admix in the Prenatal developmental study even though these doses were maternally toxic and fetal-toxic. In vitro, genistein had teratogenic potential at high concentrations in the WEC screening assay, however this was not predictive of the in vivo findings. On the basis of the definitive Prenatal development study, the NOAEL for maternal toxicity and adverse effects on embryonic development was considered to be 100 mg/kg/day when administered orally by dietary admix.     

Structure,biology, and therapeutic implications of pegylated interferon alpha-2b

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.     

Mutations of the <Emphasis Type="Italic">SDHB</Emphasis> and <Emphasis Type="Italic">SDHD</Emphasis> genes

The succinate dehydrogenase (SDH) is a mitochondrial enzyme complex with an important role in oxydative phosphorylation and intracellular oxygene sensing and signaling. Mutations in the SDHB (1p35–36) and SDHD subunits (11q23) give rise to the paraganglioma syndromes (PGL), namely PGL 4 and PGL 1, and generate paraganglioma and pheochromocytoma. For both genes mutations have been described that result in a loss of function of the gene products. SDHBmutations were found in five of eight exons and in two introns, SDHD mutations in all four exons and one intron. Phenotypes and rate of malignancy of SDHB and SDHD seem to be different, with a higher frequency of head-and-neck tumors in SDHD and indications of a higher risk of malignancy in SDHB mutations. As routine diagnostic procedure all SDH mutation carriers should have urine catecholamine analysis as well as pelvic, abdominal, thoracic and skull/neck MRI.