Novel, flexible, and conformationally defined analogs of gepirone: synthesis and 5-HT1A, 5-HT2A, and D2 receptor activity

Novel, flexible arylpiperazine gepirone analogs (1a-3a) with a mixed 5-HT1A/5-HT2A receptor profile, low D2 receptor affinity, and agonistic (2a) or partial agonistic (1a, 3a) activity toward 5-HT1A receptor sites were synthesized. Their conformationally restricted counterparts (1b-3b) were selective 5-HT1A ligands (over 5-HT2A and D2 receptors), which turned out to be agonists (2b, 3b), or partial agonist (1b) of 5-HT1A receptors.     

The role of Haldane's rule in sex allocation

Sex allocation theory predicts that parents should bias their reproductive investments toward the offspring sex generating the greatest fitness return. When females are the heterogametic sex (e.g., ZW in butterflies, some lizards, and birds), production of daughters is associated with an increased risk of offspring inviability due to the expression of paternal, detrimental recessives on the Z chromosome. Thus, daughters should primarily be produced when mating with partners of high genetic quality. When female sand lizards (Lacerta agilis) mate with genetically superior males, exhibiting high MHC Class I polymorphism, offspring sex ratios are biased towards daughters, possibly due to recruitment of more Z-carrying oocytes when females have assessed the genetic quality of their partners. If our study has general applicability across taxa, it predicts taxon-specific sex allocation effects depending on which sex is the heterogametic one.     

Functional characterization of rhodopsin monomers and dimers in detergents

Rhodopsin (Rho) is a G protein-coupled receptor that initiates phototransduction in rod photoreceptors. High expression levels of Rho in the disc membranes of rod outer segments and the propensity of Rho to form higher oligomeric structures are evident from atomic force microscopy, transmission electron microscopy, and chemical cross-linking experiments. To explore the structural and functional properties of Rho in n-dodecyl-beta-maltoside, frequently used to purify heterologously expressed Rho and its mutants, we used gel filtration techniques, blue native gel electrophoresis, and functional assays. Here, we show that in micelles containing n-dodecyl-beta-maltoside at concentrations greater than 3 mM, Rho is present as a single monomer per detergent micelle. In contrast, in 12 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), micelles contain mostly dimeric Rho. The cognate G protein transducin (Gt) appears to have a preference for binding to the Rho dimer, and the complexes fall apart in the presence of guanosine 5'-3-O-(thio)triphosphate. Cross-linked Rho dimers release the chromophore at a slower rate than monomers and are much more resistant to heat denaturation. Both Rho(*) monomers and dimers are capable of activating Gt, and both of them are phosphorylated by Rho kinase. Rho expressed in HEK293 cells is also readily cross-linked by a bifunctional reagent. These studies provide an explanation of how detergent influences the oligomer-dimermonomer equilibrium of Rho and describe the functional characterization of Rho monomers and dimers in detergent.     

Effect of early thermal experience on pituitary-gonadal axis in female rats

The reproductive system fully develops during postnatal stages of life, and as such it may be susceptible to environmental cues such as high temperature. Thus, the purpose of the study was to compare how exposure to 34 degrees Celsius ambient temperature affects pituitary-gonadal axis of immature and adult female rats. Three groups of females at proestrous or metaestrous (n=38) were used in the study. The females were housed in ambient temperature of 34 degrees Celsius (WR group, n=10) or room temperature (CR group, n=16) from birth to adulthood. The females from the third group were acclimated to 34 degrees Celsius as adults (WA group, n=12). In the WR group the onset of puberty was delayed in comparison to the CR group. The plasma PRL level was lower during proestrous and higher during metaestrous in the WR group compared to the CR group. During metaestrous, lower FSH and higher progesterone (P(4)) plasma concentrations were found in the WR females. No changes in LH and oestradiol (E(2)) plasma concentrations were demonstrated. Higher in vitro E(2) and lower P(4) secretions under FSH stimulation were observed in the WR follicles compared to those of the CR group. The WR group also demonstrated higher basal and LH-stimulated luteal in vitro secretion of P(4) than controls. Plasma LH and FSH concentrations during metaestrous were higher in WA females than in the WR group, but PRL level was lower. Follicles of the WA group were unresponsive to FSH with respect to steroid secretion. In addition, LH stimulated luteal E(2) secretion in this group. P(4) release by luteal cells was lower in the WA than in the WR group. We concluded that WR females differ from WA in reproductive system adjustments to rearing temperature and that early thermal experience is more effective in antagonizing the effect of high temperature than acclimation of adult females.     

Expression of the selected adhesive molecules (cadherin E, CD44, LGAL3 and CA50) in papillary thyroid carcinoma

INTRODUCTION: The aim of the study was to determine the expression of selected adhesive molecules in papillary thyroid carcinoma. MATERIAL AND METHODS: 47 papillary thyroid carcinoma cases and 11 nonmalignant goiter cases were analyzed by immunohistochemistry. RESULTS: Galectin-3 (LGAL3) was a sensitive and specific marker, present in 91% of analyzed tumors and only in 5% of tumor margin. The presence of CA50 was 86% and 3% respectively with only 3% positive non-malignant cases. Cadherin E expression was noted in 91% of primary tumors, in 84% of the surrounding tissue and in 63% of non-malignant goiter. CD44 (DF1485) was observed in 89% of primary tumors and 48% of surrounding tissue; the reaction with BBA10 was more characteristic for metastases. CONCLUSIONS: Our study confirms the high diagnostic value of galectin-3 in papillary thyroid carcinoma and reveals the similar efficiency of CA50. CD44 (DF1485) expression in primary tumor is more intensive than in surrounding tissue, but the diagnostical inportance is not high because it is often observed in benign lesions. Using of BBA10 is more sensitive, but less specific. High expression of cadherin E in benign lesions impairs its diagnostical application in papillary thyroid cancer.     

A special case of thyroid associated ophthalmopathy in the course of Graves-Basedow disease

INTRODUCTION: The exact pathogenesis of Graves' ophthalmopathy and the possibility of causal treatment of this disease still remain unclear. Currently no standard treatment guidelines have been accepted. While treatment procedures have been established in specialized centres, management of complicated and long-lasting cases is always individual. We present an unusual case of Graves' ophthalmopathy accompanied by other autoimmune diseases. CASE REPORT: Our patient, MB, female, born in 1961, was diagnosed with Graves' disease 13 years ago. Recurrent hyperthyroidism and large goitre qualified her for strumectomy (performed twice) and long-term antithyroid treatment. Four years after her initial diagnosis, relapsing severe (ophthalmopathy index: 9 points, CAS: 7 points) occurred which persisted despite continuous administration of glucocorticoids. Due to imminent blindness, orbital decompression had to be performed, three times since. Concurrent autoimmune diseases: ulcerative colitis and seronegative rheumatoid arthritis were also stated. Two years ago, due to loss of vision acuity, rapid progression of exophthalmos and recurrence of hyperthyroidism, immunosuppressive treatment with azathioprine was undertaken over a period of 12 months. The present condition of the patient is satisfactory. CONCLUSION: Judging from the discussed course of treatment, in rare and difficult cases of proliferative ophthalmopathy, early immunosuppressive treatment other than glucocorticoids, should be considered.     

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

The Oxidative Stress May be Induced by the Elevated Homocysteine in Schizophrenic Patients

The mechanisms of oxidative stress in schizophrenic patients are not fully understood. In the present study, we investigated the effect of elevated level of homocysteine (Hcys) on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in plasma, the level of carbonyl groups in plasma proteins, as well as the amount of 3-nitrotyrosine in plasma proteins isolated from schizophrenic patients. Patients hospitalised in I and II Psychiatric Department of Medical University in Lodz, Poland were interviewed with special questionnaire (treatment, course of diseases, dyskinesis and other EPS). According to DSM-IV criteria all patients had diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). Mean time of schizophrenia duration was about 5 years. High-performance liquid chromatography was used to analyse the total level of homocysteine in plasma. Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. The lipid peroxidation in plasma was measured by the level of TBARS. Our results showed that in schizophrenic patients the amount of homocysteine in plasma was higher in comparison with the control group. We also observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. Moreover, our experiments indicate that the correlation between the increased amount of homocysteine and the oxidative stress exists. Considering the data presented in this study, we suggest that the elevated Hcys in schizophrenic patients may stimulate the oxidative stress.     

Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.