Andrej Nikoshkov

Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers.

μ Opioid receptors are critical for heroin dependence, and A118G SNP of the μ opioid receptor gene (OPRM1) has been linked with heroin abuse. In our population of European Caucasians (n = 118), ≈90% of 118G allelic carriers were heroin users. Postmortem brain analyses showed the OPRM1 genotype associated with transcription, translation, and processing of the human striatal opioid neuropeptide system. Whereas down-regulation of preproenkephalin and preprodynorphin genes was evident in all heroin users, the effects were exaggerated in 118G subjects and were most prominent for preproenkephalin in the nucleus accumbens shell. Reduced opioid neuropeptide transcription was accompanied by increased dynorphin and enkephalin peptide concentrations exclusively in 118G heroin subjects, suggesting that the peptide processing is associated with the OPRM1 genotype. Abnormal gene expression related to peptide convertase and ubiquitin/proteosome regulation was also evident in heroin users. Taken together, alterations in opioid neuropeptide systems might underlie enhanced opiate abuse vulnerability apparent in 118G individuals.

Opioid neuropeptide genotypes in relation to heroin abuse: Dopamine tone contributes to reversed mesolimbic proenkephalin expression

Striatal enkephalin and dynorphin opioid systems mediate reward and negative affect, respectively, relevant to addiction disorders. We examined polymorphisms of proenkephalin (PENK) and prodynorphin (PDYN) genes in relation to heroin abuse and gene expression in the human striatum and the relevance of genetic dopaminergic tone, critical for drug reward and striatal function. Heroin abuse was significantly associated with PENK polymorphic 3′ UTR dinucleotide (CA) repeats; 79% of subjects homozygous for the 79-bp allele were heroin abusers. Such individuals tended to express higher PENK mRNA than the 81-bp homozygotes, but PENK levels within the nucleus accumbens (NAc) shell were most strongly correlated to catecholamine-O-methyltransferase (COMT) genotype. Control Met/Met individuals expressed lower PENK mRNA than Val carriers, a pattern reversed in heroin users. Up-regulation of NAc PENK in Met/Met heroin abusers was accompanied by impaired tyrosine hydroxylase (TH) mRNA expression in mesolimbic dopamine neurons. In contrast to PENK, no association was detected between PDYN genotype (68-bp repeat element containing one to four copies of AP-1 binding sites in the promoter region) and heroin abuse, although there was a clear functional association with striatal PDYN mRNA expression: an increased number of inducible repeats (three and four) correlated with higher PDYN levels than adult or fetal subjects with noninducible (one and two) alleles. Moreover, PDYN expression was not related to COMT genotype. Altogether, the data suggest that dysfunction of the opioid reward system is significantly linked to opiate abuse vulnerability and that heroin use alters the apparent influence of heritable dopamine tone on mesolimbic PENK and TH function.

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction

Abstract Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues.

Naturally Occurring Mutants of Human Steroid 21-Hydroxylase (P450c21) Pinpoint Residues Important for Enzyme Activity and Stability

Three mutants (deletion of E196, G291S, and R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited congenital adrenal hyperplasia had reduced activity toward progesterone and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the half-life of the G291S mutant was comparable with that of the normal protein. These results directly link the clinical situation with the three mutations and suggest that G291 is important for the catalytic activity of P450c21.

Prodynorphin transcripts and proteins differentially expressed and regulated in the adult human brain

Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell‐specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions and are implicated in various neuropsychiatric disorders. Here, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Seven PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full‐length PDYN. The dominant, FL1 transcript shows high expression in limbic‐related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and hypothalamus. FL‐PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N‐terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system.

Response to Treatment in Patients with Partial Androgen Insensitivity due to Mutations in the DNA-Binding Domain of the Androgen Receptor

The androgen insensitivity syndrome is a disorder caused by deficient function of the androgen receptor, characterized by varying degrees of undermasculinization in karyotypic males. We have identified four mutations in the androgen receptor gene, in the region encoding the DNA-binding domain of the protein. Two mutations, R607X and R615G, were found in patients with complete insensitivity to androgens, whereas the other two, S578T and A596T, were found in patients with partial insensitivity. The functional consequences of the three missense mutations were assayed in vitro after transient expression of the receptors in COS cells. All mutants showed normal androgen binding but abnormal abilities to stimulate transcription of an androgen-responsive reporter gene. R615G abolished transactivation whereas S578T and A596T were partially malfunctional. The function of A596T, but not of S578T, was normalized at high androgen concentrations in vitro, reflecting the in vivo situation. Thus, patients with specific mutations in the DNA-binding domain of the androgen receptor may benefit from androgen treatment.

Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid.

Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice

We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.

A novel mutation (N233K) in the transactivating domain and the N756S mutation in the ligand binding domain of the androgen receptor gene are associated with male infertility

OBJECTIVE Resistance to androgens has been suggested as a possible cause of male infertility. This hypothesis is based mainly on binding studies in genital skin fibroblasts but the molecular evidence is sparse.

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [3H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.