Radovan Komel

Association of BDNF with restricting anorexia nervosa and minimum body mass index: a family-based association study of eight European populations

Eating disorders (ED), such as anorexia nervosa (AN) and bulimia nervosa (BN), are complex psychiatric disorders where different genetic and environmental factors are involved. Several lines of evidence support that brain-derived neurotrophic factor (BDNF) plays an essential role in eating behaviour and that alterations on this neurotrophic system participates in the susceptibility to both AN and BN. Accordingly, intraventricular administration of BDNF in rats determines food starvation and body weight loss, while BDNF or its specific receptor NTRK2 knockout mice develop obesity and hyperphagia. Case–control studies also suggest a BDNF contribution in the aetiology of ED: we have previously reported a strong association between the Met66 variant within the BDNF gene, restricting AN (ANR) and minimum body mass index (minBMI) in a Spanish sample, and a positive association between the Val66Met and −270C/T BDNF SNPs and ED in six different European populations. To replicate these results, avoiding population stratification effects, we recruited 453 ED trios from eight European centres and performed a family-based association study. Both haplotype relative risk (HRR) and haplotype-based haplotype relative risk (HHRR) methods showed a positive association between the Met66 allele and ANR. Consistently, we also observed an effect of the Met66 variant on low minBMI and a preferential transmission of the −270C/Met66 haplotype to the affected ANR offspring. These results support the involvement of BDNF in eating behaviour and further suggest its participation in the genetic susceptibility to ED, mainly ANR and low minBMI.

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance.

The nucleotide sequence of the Bacillus licheniformis bacitracin‐resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP‐binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro‐phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram‐negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide‐binding sites, BcrA protein and mammalian multidrug transporter or P‐glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide‐binding protein to determine the Importance of various regions in transport.

Engineering inclusion bodies for non denaturing extraction of functional proteins

BackgroundFor a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-α (Truncated form of Tumor necrosis factor α with an N-terminal histidine tag) and dN19 LT-α (Truncated form of Lymphotoxin α).ResultsFour structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein.Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7ΔN6TNF-α, and GFP, a significant amount of protein could be extracted from IBs with 0.2% N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied.ConclusionThis study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals.

The association between brain-derived neurotrophic factor polymorphism (BDNF Val66Met) and suicide

BACKGROUND Brain-derived neurotrophic factor (BDNF) mediates neural plasticity, mood, different behaviours, and stress response. A functional BDNF polymorphism (BDNF Val66Met) was reported to influence the effects of stressful life events or childhood adversity on depression and suicidal behaviour in various psychopathologies. The study evaluated the association between BDNF Val66Met variants and suicide, committed with violent or non-violent methods, in victims with or without stressful childhood experience. METHODS BDNF Val66Met polymorphism was genotyped on 560DNA samples from 359 suicide victims and 201 control subjects collected on autopsy from unrelated Caucasian subjects and subdivided according to gender, method of suicide, and influence of childhood adversity. RESULTS A similar frequency of BDNF Val66Met variants was found between all included suicide victims and the control groups, and also between the male groups. The frequency of the combined Met/Met and Met/Val genotypes and the homozygous Val/Val genotype was significantly different between the female suicide victims and female controls, between the female suicide victims who used violent suicide methods and female controls, and between all included suicide victims with or without stressful life events. The combined Met/Met and Met/Val genotypes contributed to this significance. LIMITATION A small group of suicide victims with available data on childhood adversity was studied. CONCLUSIONS The combined Met/Met and Met/Val genotypes of the BDNF Val66Met variant could be the risk factor for violent suicide in female subjects and for suicide in victims exposed to childhood trauma. These results confirm a major role of BDNF in increased vulnerability to suicide.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

BackgroundIn recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process.To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed.ResultsIn this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared.During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation.During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity.High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity.ConclusionsThe study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

VDR Genotype and Response to Etidronate Therapy in Late Postmenopausal Women

Abstract: Twenty-four late postmenopausal women with osteoporosis were studied. The patients were separated in three subgroups according to the BsmI polymorphism of the vitamin D receptor (VDR) gene: BB (n= 8), Bb (n = 10) and bb (n = 6). They did not differ in age (mean ages were 66.0 years, 65.9 years and 63.9 years, respectively), years after menopause (18.7 years, 18.1 years and 18.4 years) or body weight (64.9 kg, 65.3 kg and 63.8 kg), the variables known to be associated with bone mineral density (BMD). The results show that the response to antiresorptive bisphosphonate therapy in combination with calcium supplementation is modified by VDR genotype. The lumbar spine BMD increased significantly faster in the BB and Bb groups (7.3% and 7.0%, respectively) compared with the bb group (2.5%) during 1 year of cyclic etidronate therapy (400 mg/day) and calcium supplementation (1000 mg/day). The biochemical marker of bone resorption (urinary hydroxyproline excretion) as well as the bone formation marker (serum levels of osteocalcin) decreased during the treatment. With respect to VDR genotype, a significantly higher decrease in osteocalcin level was observed in bb as compared with BB subjects. We conclude that the VDR genotype is involved in an individual’s response to cyclic etidronate therapy with calcium supplementation.

Combined family trio and case-control analysis of the COMT Val158Met polymorphism in European patients with anorexia nervosa.

The high activity Val158 (H) allele of the dopamine‐metabolizing enzyme catechol‐O‐methyltransferase (COMT) was associated with anorexia nervosa (AN) in a recent family trio‐based study of patients from Israel. In an attempt to replicate this finding, we performed a combined family trio and case‐control study in an European population from seven centers in six different countries (Austria, Germany, Great Britain, Italy [Milan], Italy [Florence], Slovenia, and Spain), together contributing a total of 372 family trios, 684 controls and 266 cases. TDT analyses of high (H) and low (L) alleles in family trios showed that H allele and L allele were each transmitted 101 times (χ2 = 0, ns). Allele‐wise case‐control analysis using separate samples simply combined from the centers was also not significant, with the frequencies of the H allele 50% in cases and same in controls. Stratified analysis of data from all centers gave an odds ratio of 0.98 (Cornfield 95% confidence limits 0.78–1.24). Analysis by genotype was likewise not significant (overall χ2 = 0.42). Because we were not able to support the primary hypothesis that Val158Met is a risk factor for AN, we did not perform secondary analysis of minimum body mass index (mBMI), age at onset or illness subtype (restricting or binge purging anorexia). Overall we found no support for the hypothesis that the Val158 allele of COMT gene is associated with AN in our combined European sample.

Analysis of 14 cystic fibrosis mutations in five South European populations

SummaryWe have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugoslavian) for 14 cystic fibrosis (CF) mutations. The most frequent mutations, apart from ΔF508, were G542X (6.04%), R1162X (3.61%) and N1303K (3.24%). Each of the other analysed mutations were present at a frequency of less than 1% (R347P, R334W, S549RA, S549I, G551D, R553X and W1282X), and four mutations (D110H, ΔI507, S549RT, and S1255X) were not found in this sample. The data presented here allows the use of mutation analysis in 69.5% of Spanish, 58% of Greek, and 56.5% of Italian CF cases.

No major effect of estrogen receptor beta gene RsaI polymorphism on bone mineral density and response to alendronate therapy in postmenopausal osteoporosis

Genetic factors play an important role in the pathogenesis of osteoporosis. The genes involved are, however, still largely unknown. In the present study, we have investigated whether sequence variations in the estrogen receptor beta (ERbeta) gene are associated with bone mineral density (BMD) and biochemical markers of bone turnover in 79 Slovenian postmenopausal women with osteoporosis. We also assessed the response by BMD and bone markers to antiresorptive therapy with bisphosphonate alendronate. All eight exons of ERbeta gene were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis. Potentially mutated samples were found only in exon 5 and sequence analysis identified the presence of a silent mutation in codon 328 with a nucleotide substitution GTG to GTA. For easier detection of this silent mutation, the RsaI restriction fragment length polymorphism analysis was developed. The frequencies of genotypes were as follows: Rr 5.1% and RR 94.9%. Between both genotypes, no significant differences in baseline lumbar spine and femoral neck BMD or in bone markers osteocalcin and deoxypyridinoline were observed. Similarly, no significant difference between RR and Rr genotypes in BMD or bone markers after 1 year of therapy was found. The increase in lumbar spine BMD after therapy was the only parameter that approached statistical significance (P=0.099). Patients with genotype Rr showed a smaller increase compared to those with RR. Our results suggest that RsaI polymorphism of ERbeta gene is probably not an important genetic determinant of BMD and does not significantly influence the responsiveness to alendronate therapy.

Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets.

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca2+ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca2+ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration.