MDMA (3,4-Methylenedioxymethamphetamine) Analogues as Tools to Characterize MDMA-Like Effects: An Approach to Understand Entactogen Pharmacology.

Besides stimulants and hallucinogens, whose psychotropic effects are shared by many structurally related molecules exhibiting different efficacies and potencies in humans, the phenylisopropylamine MDMA (3,4-methylenedioxymethamphetamine, XTC, "Ecstasy") is the prototypical representative of a separate class of psychotropic substance, able to elicit the so-called entactogenic syndrome in healthy humans. This reversible altered state of consciousness, usually described as an "open mind state", may have relevant therapeutic applications, both in psychotherapy and as a pharmacological support in many neuropsychiatric disorders with a high rate of treatment failure. Nevertheless, a comprehensive and systematic exploration of the structure-activity relationships associated with entactogenic activity has remained incomplete and controversial, highlighting the possibility that MDMA might represent a pharmacological rarity in the field of psychotropics. As the latter is still an open question, the pharmacological characterization of MDMA analogues remains the logical strategy to attempt the elucidation of the structural requirements needed to elicit typical MDMA-like effects. Intriguingly, almost no experimental evidence supports the existence of actual MDMA analogues that truly resemble the whole pharmacological profile of MDMA, probably due to its complex (and partially not fully understood) mechanism of action that includes a disruption of monoaminergic neurotransmission. The present review presents a brief summary of the pharmacology of MDMA, followed by the evidence accumulated over the years regarding the characterization of classical structurally related MDMA analogues in different models and how this state of the art highlights the need to develop new and better MDMA analogues.

Mild clinical and biochemical phenotype in two patients with PMM2-CDG (congenital disorder of glycosylation Ia).

Phosphomannomutase 2 deficiency (PMM2-CDG) patients may present as mild phenotypes, with the cerebellum frequently involved. In those cases, false-negative results in screening may occur when applying conventional biochemical procedures. Our aim was to report two patients with a diagnosis of PMM2-CDG presenting with mild clinical phenotype. Patient 1-at 9 months of age, she presented with just psychomotor delay, tremor, hypotonia, and slight lipodystrophy. Patient 2-she presented at 8 months of age with psychomotor delay, hand stereotypes, hypotonia, convergent bilateral strabismus, and tremor but no lipodystrophy. Routine biochemical parameters including blood count, clotting factors, proteins, and thyroid hormone were normal in both cases. Cranial MRI evidenced mild cerebellar atrophy with moderate vermis hypoplasia. In case 1, sialotransferrin pattern showed very slightly increased disialotransferrin with no asialotransferrin, and in case 2, the transferrin pattern was impaired in the first study but nearly normal in the second. Nevertheless, in all the samples, quantification of the patterns obtained by capillary zone electrophoresis analysis gave results out of the control range. High residual PMM2 activity was observed in both cases and the genetic analysis showed that patient 1 was heterozygous for c.722G>C (p.C241S) and c.368G>A (p.R123Q) mutations, and patient 2 showed the c.722G>C and the c.470T>C (p.F157S) mutations in the PMM2 gene. We would like to stress the importance of the use of sensitive semiquantitative methods of screening for CDG in order to achieve early identification of patients with mild phenotypes. Intentional tremor was an atypical but remarkable clinical feature in both cases, and the global cerebellar atrophy with vermis hypoplasia reinforced the early clinical suspicion of a PMM2-CDG disease.

Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency.

We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.

The role of human demographic history in determining the distribution and frequency of transferase-deficient galactosaemia mutations.

Classical or transferase-deficient galactosaemia is an inherited metabolic disorder caused by mutation in the human Galactose-1-phosphate uridyl transferase (GALT) gene. Of some 170 causative mutations reported, fewer than 10% are observed in more than one geographic region or ethnic group. To better understand the population history of the common GALT mutations, we have established a haplotyping system for the GALT locus incorporating eight single nucleotide polymorphisms and three short tandem repeat markers. We analysed haplotypes associated with the three most frequent GALT gene mutations, Q188R, K285N and Duarte-2 (D2), and estimated their age. Haplotype diversity, in conjunction with measures of genetic diversity and of linkage disequilibrium, indicated that Q188R and K285N are European mutations. The Q188R mutation arose in central Europe within the last 20 000 years, with its observed east-west cline of increasing relative allele frequency possibly being due to population expansion during the re-colonization of Europe by Homo sapiens in the Mesolithic age. K285N was found to be a younger mutation that originated in Eastern Europe and is probably more geographically restricted as it arose after all major European population expansions. The D2 variant was found to be an ancient mutation that originated before the expansion of Homo sapiens out of Africa.

Secondary disorders of glycosylation in inborn errors of fructose metabolism.

Adamowicz and colleagues raised the alert in 2007 about patients with atypical hereditary fructose intolerance (HFI) primarily misdiagnosed as CDG Ix. We describe a girl with neonatal hypertonia, facial trismus, absent swallowing and coughing reflexes, gastro-oesophageal reflux and sporadically elevated Krebs cycle metabolites and lactate. At 14 months microcephaly and hepatomegaly were noted, with hypertransaminasaemia but normal blood coagulation, glucose, phosphate, and absent urinary reducing substances. Neurological impairment persisted. Because of hepatic and neurological abnormalities with developmental delay, Tf IEF was performed and showed a severe type 1 pattern, resulting in a wrong diagnosis of CDG. Subsequently, an aversion to fruits suggested HFI, confirmed by the finding of ALDOB mutations (p.A150P/p.N335K). The girl improved with fructose-free diet, but liver cirrhosis led to hepatic transplantation. She is now 7 years old with good evolution; facial trismus and hypertonia reversed, but microcephaly persists. Transferrin MALDI-TOF MS characterization revealed underoccupation of glycosylation sites and glycan abnormalities, which reversed with dietary treatment. High maternal fructose concentrations might have caused neonatal abnormalities. Although in our patient's mother there is no fructose accumulation at present, it is possible that increased ingestion of fruits and vegetables during pregnancy, together with her heterozygosity, caused an accumulation of fructose that finally affected the fetus. We also describe slightly abnormal transferrin isoelectric focusing and MALDI-TOF MS patterns of intact transferrin and N-glycans in a fructose-1,6-bisphosphatase (FBP1)-deficient patient. While HFI is a well-known cause of secondary CDG, we found no reports of abnormal transferrin isoelectric focusing patterns in FBP1 deficiency and we introduce this condition as a possible secondary cause for altered transferrin isoelectric focusing.

PDH E1ß deficiency with novel mutations in two patients with Leigh syndrome.

Most cases of pyruvate dehydrogenase complex (PDHc) deficiency are attributable to mutations in the PDHA1 gene which encodes the E(1)α subunit, with few cases of mutations in the genes for E(3), E3BP (E(3) binding protein), E(2) and E(1)-phosphatase being reported. Only seven patients with deficiency of the E(1)ß subunit have been described, with mutations in the PDHB gene in six of them. Clinically they presented with a non-specific encephalomyopathy. We report two patients with new mutations in PDHB and Leigh syndrome. Patient 1 was a boy with neonatal onset of hyperlactataemia, corpus callosum hypoplasia and a convulsive encephalopathy. After neurological deterioration, he died at age 5 months. Autopsy revealed the characteristic features of Leigh syndrome. Patient 2, also a boy, presented a milder clinical course. First symptoms were noticed at age 16 months with muscular hypotonia, lactic acidosis and recurrent episodes of somnolence and transient tetraparesis. MRI revealed bilateral signal hyperintensities in the globus pallidus, midbrain and crura cerebri. PDHc and E(1) activities were deficient in fibroblasts in patient 1; in patient 2 PDHc deficiency was found in skeletal muscle. Mutations in PDHA1 were excluded. Sequencing of PDHB revealed a homozygous point mutation (c.302T>C), causing a predicted amino acid change (p.M101T) in patient 1. Patient 2 is compound heterozygote for mutations c.301A>G (p.M101V) and c.313G>A (p.R105Q). All three mutations appear to destabilize the E(1) enzyme with a decrease of both E(1)α and E(1)ß subunits in immunoblot analysis. To our knowledge, these patients with novel PDHB mutations are the first reported with Leigh syndrome.

[Infection-triggered familial or recurrent acute necrotizing encephalopathy]. / Encefalopatía aguda necrosante familiar o recurrente desencadenada por infecciones.

Acute necrotizing encephalopathy (ANE) presents in children after common viral infections. Most cases of ANE are non-familial and non-recurrent and have been mainly reported in Asian patients, although ANE affects children worldwide. Recently, missense mutations in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2) have been found in several families with familial or recurrent cases of ANE. We describe a Spanish family with familial and recurrent ANE without mutations in RANBP2. Mutations in RANBP2 are not the sole susceptibility alleles for familial or recurrent ANE.

Secondary alteration of the transferrin isoelectric focusing pattern in a case of bacterial meningitis.

Congenital disorders of glycosylation (CDG) are a group of inherited defects in the synthesis and processing of the linked glycans of glycoproteins and other glycoconjugates. The phenotypic spectrum presents wide variability, and clinical diagnosis is not reliable in most cases. Isoelectric focusing (IEF) of serum transferrin is widely used as a tool to detect CDG. We describe a paediatric patient presenting an altered serum transferrin pattern due to a secondary disorder of glycosylation caused by pneumococcal meningitis (Streptococcus pneumoniae, serotype 19A). During admission, brain CT scan and MRI showed acute ischaemic lesions in brain frontotemporal parenchyma, and enlarged subarachnoidal spaces in the frontal area resembling a chronic injury. This led us to screen for inborn errors of metabolism potentially associated with these findings (homocystinuria, glutaric aciduria, CDG syndromes). Biochemical studies for the screening of these inborn errors of metabolism were normal except for sialotransferrin isoelectric focusing, which showed a type 2 pattern. However, 16 days later, together with the remission of the meningitis process, the sialotransferrin pattern had normalized. The apolipoprotein C-III (an O-glycoprotein) profile was normal in all samples analysed. In conclusion, infectious events should be ruled out in the differential diagnosis of CDG syndromes. Furthermore, our findings highlight the possibility that the type 2 IEF pattern of serum sialotransferrin detected in some patients with neonatal death due to organ failure and septic events might be secondary to the infectious process.

Mutational spectrum of classical galactosaemia in Spain and Portugal.

Classical galactosaemia is an autosomal recessive inherited metabolic disorder due to deficient galactose-1-phosphate uridyltransferase (GALT). Over 180 different base changes and disease-causing mutations have been reported in the GALT gene. Mutation p.Q188R was found to be the most common molecular defect among caucasian classical galactosaemia patients. We have characterized the spectrum of GALT mutations in a group of 51 Spanish families and 32 Portuguese families with this disease. p.Q188R is also the most prevalent mutation in the Spanish and Portuguese population, accounting for 50% and 57.8% of galactosaemic alleles, respectively. An additional 15 mutations were also identified in Spanish patients, four of which were novel: p.D28H, p.S181A, c.658dupG and c.377+53_1059+87del. In the Portuguese population, 11 different mutations were found, three of which were novel: p.R33H, p.P185S, and p.S192G. The differences observed between the genotypes identified in Portuguese and Spanish galactosaemic populations are notable. Only mutations p.Q188R, p.R148Q and c.820+13g>a were identified in both populations. In spite of the geographical proximity of Spain and Portugal, it seems that they have received genetic influences from different populations. The repeated migrations that occurred in the Iberian Peninsula throughout centuries may explain such variability.

The endonuclease NL1Tc encoded by the LINE L1Tc from Trypanosoma cruzi protects parasites from daunorubicin DNA damage.

In the present paper we show that the overexpression of the NL1Tc protein, encoded by the L1Tc non-LTR retrotransposon from Trypanosoma cruzi, led to a reduction of about 60% of DNA damage caused by daunorubicin treatment. This repair effect is not observed in transfected parasites overexpressing the NL1Tc mutated in the aspartic acid located in the active site of the enzyme. In addition, NL1Tc overexpression protects the parasite from the negative effect that daunorubicin has on parasite's growth rate. Thus, parasites overexpressing NL1Tc show, after treatment with 4 microM of daunorubicin, growth rate two to three times higher than the growth rate observed in treated control parasites transformed with the empty vector or overexpressing the mutated NL1Tc. Likewise, parasites overexpressing the NL1Tc protein and irradiated with a single dose of gamma-radiation (6 or 9 Gy) show higher growth rates than the parasites overexpressing the mutated NL1Tc or the control transfected parasites.

Muscle coenzyme Q10 concentrations in patients with probable and definite diagnosis of respiratory chain disorders.

Coenzyme Q(10) (CoQ) deficiency syndrome is a disorder of unknown ethiology that may cause different forms of mitochondrial encephalomyopathy. In the present study our aim was to analyse CoQ concentration and mitochondrial respiratory chain (MRC) enzyme activities in muscle biopsies of patients with clinical suspicion and/or biochemical-molecular diagnosis of a mitochondrial disorder. We studied 36 patients classified into 3 groups: 1) 14 patients without a definitive diagnosis of mitochondrial disease, 2) 13 patients with decreased CI + III and II + III activities of the MRC, and 3) 9 patients with definitive diagnosis of mitochondrial disease. Only 1 of the 14 patients of group 1 showed slightly reduced CoQ values in muscle. Six of the 13 patients from group 2 showed partial CoQ deficiency in muscle and 1 of the 9 cases from group 3 presented a slight CoQ deficiency. Significantly positive correlation was observed between CI + III and CII + III activities with CoQ concentrations in the 36 muscle homogenates from patients (r = 0.555; p = 0.001; and r = 0.460; p = 0.005, respectively). In conclusion, measurement of MRC enzyme activities is a useful tool for the detection of CoQ deficiency, which should be confirmed by CoQ quantification.

[Treatment response to lamivudine in chronic hepatitis B patients]. / Respuesta al tratamiento con lamivudina en pacientes con hepatitis crónica por virus B.

OBJECTIVE: The main objective was to find a biochemistry and virology response in positive and negative HBeAg patients who had been treating with lamivudine. Furthermore, the secondary objective was to make a description about kidney function changes that could be found in patients under treatment with lamivudine. METHOD: Thirty patients with chronic B hepatitis under treatment with lamivudine had been retrospectively studied since November 1999 to March 2004, in a Digestive Unit, in Ciudad Real Hospital. RESULTS: Twenty nine out of 30 patients were included, 8 out of 29 were positive HBeAg, and 21 out of 29 were negative HBeAg. Seven patients had been treated with IFN. Thus, 8 patients had been in need of adefovir treatment after lamivudine trial. The average of treatment had been 23.7 months (3 months-46 months). In fact, none of them were changed from positive HBsAg to negative HBsAg. Nevertheless, we have observed those results after lamivudine treatment. Firstly, 50% positive HBe Ag patients have carried on negative DNA results. Although 25% positive HBe Ag patients had converted to negative HBe Ag. But they did not show any anti HBe. Secondly, 76% negative HBe Ag patients had been suffering a DNA decreased, even though some of them had had a negative DNA results. Thirdly, we found some lamivudine mutation (50% in positive HBe Ag patients and 19% in negative HBe Ag patients). Finally, all the patients with negative DNA had maintained a normal AST and ALT levels. Thus, function renal had been normal under lamivudine trial. CONCLUSION: According to several authors, the response to lamivudine treatment had been adequate in our population. Nevertheless, our study have shown that antiHBe levels had not been suffering any change during lamivudine treatment. In addition, we have found a smaller figures (19%) of lamivudine mutations in negative HBe Ag patients than other studies (25% in patients who were followed up for 1 year).

Methylphenidate has long-lasting metaplastic effects in the prefrontal cortex of adolescent rats.

Methylphenidate (MPH) is widely used as a "nootropic" agent and in the treatment of disorders of attention, and has been shown to modulate synaptic plasticity in vitro. Here we present in vivo evidence that this MPH-induced metaplasticity can last long after the end of treatment. MPH (0, 0.2, 1 and 5mg/kg) was administered daily to male rats from postnatal day 42 for 15 days. The animals were tested daily in a radial maze. Long-term potentiation (LTP), a marker of neural plasticity, was induced in vivo in the prefrontal cortex after 2-3h, 15-18 days or 5 months without treatment. The behavioral performance of the 1mg/kg group improved, while that of animals that had received 5mg/kg deteriorated. In the 1 and 5mg/kg groups LTP induced 2-3h after the last MPH treatment was twice as large as in the controls. Further, 15-18 days after the last MPH administration, in groups receiving 1 and 5mg/kg, LTP was about fourfold higher than in controls. However, 5 months later, LTP in the 1mg/kg group was similar to controls and in the 5mg/kg group LTP could not be induced at all. No significant changes of LTP were seen in the low-dose group of animals (0.2mg/kg). Thus, firstly, doses of MPH that improve learning coincide approximately with those that augment LTP. Secondly, MPH-induced increases in LTP can last for several weeks, but these may disappear over longer periods or deteriorate at high doses.

Potential relationship between genotype and clinical outcome in propionic acidaemia patients.

Propionic acidaemia (PA) is an autosomal recessive disorder caused by mutations in either of the PCCA or PCCB genes which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). In this work we have examined the biochemical findings and clinical outcome of 37 Spanish PA patients in relation to the mutations found in both PCCA and PCCB genes. We have detected 27 early-onset and 101 late-onset cases, showing remarkably similar biochemical features without relation to either the age of onset of the disease or the defective gene they have. Twenty-one of the patients have so far survived and three of them, now adolescents, present normal development. Different biochemical procedures allowed us to identify the defective gene in 9 PCCA deficient and 28 PCCB deficient patients. Nine putative disease-causing mutations accounting for 77.7% of mutant alleles were identified among PCCA deficient patients, each one carrying a unique genotypic combination. Of PCCB mutant alleles 98% were characterised. Four common mutations (ins/del, E168K, 1170insT and A497V) were found in 38/52 mutant chromosomes investigated, whereas the remainder of the alleles harbour 12 other different mutations. By examining the mutations identified both in PCCA and PCCB genes and the clinical evolution of patients, we have found a good correlation between certain mutations which can be considered as null with a severe phenotype, while certain missense mutations tend to be related to the late and mild forms of the disease. Expression studies, particularly of the missense mutations identified are necessary but other genetic and environmental factors probably contribute to the phenotypic variability observed in PA.

Experimental design: a useful tool for PCR Optimization.

Because of complex interactions among the components of PCR and the wide and increasing variety of applications in which this technique is used, optimization is necessary for every reaction. Here we describe the use of experimental design techniques (2k fractional factorial design and central composite design) to attain easier, quicker and cheaper PCR optimization of DNA from blood spots. By determining the factors affecting the product yield first (factors screening), the quantity of template DNA needed for PCR and the Mg2+ concentration are easily optimized (factors optimization).

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Morphology of the mitochondria in heat shock protein 60 deficient fibroblasts from mitochondrial myopathy patients. Effects of stress conditions.

We have described two mitochondrial (mt) myopathy patients with reduced activities of various mt enzymes associated with significantly decreased amounts of heat shock protein 60 (hsp60). Experimental evidence suggested that the lack of hsp60 was the primary defect. Since hsp60 is essential for the proper folding of enzyme subunits in the mt matrix a partial deficiency of this protein can explain the observed defects of the mitochondria. Here we report on morphological studies aimed at obtaining more insight into the relation between lack of hsp60 and pathological changes of the mitochondria. Under standard culture conditions mitochondria in the partially hsp60 deficient fibroblasts showed profound morphological aberrations. In contrast, the mitochondria in fibroblasts from a MELAS patient and a cytochrome c oxidase-deficient patient appeared normal. Under stress conditions the integrity of the hsp60 deficient mitochondria declined even further: heat shock induced a temporary collapse of the electrochemical potential across the inner mt membrane, but did not affect the ultrastructure of the mitochondria; prolonged growth in confluent cultures resulted in decrease in mt number. The altered mt morphology in the hsp60 deficient cells is probably indicative of the severely impaired mt metabolism whereas the decreased stress tolerance is likely to be a direct result of paucity of the heat shock protein. Both variables are potentially useful in the diagnosis and molecular characterization of mt disorders with systemic manifestation and multiple enzyme deficiency.