Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.     

Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A(2) enzymes

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).     

A nuclear 3'-5' exonuclease proofreads for the exonuclease-deficient DNA polymerase alpha

DNA replication is a highly accurate process designed to duplicate the entire genome of a cell during each cell division. The accuracy of DNA replication is derived from the balance between three important components: base selectivity by the replicative DNA polymerases (pols), exonucleolytic proofreading, and post-replicative mismatch repair. Previously we identified a human 3'-5' exonuclease (exoN) whose properties suggested it may function as a proofreader for the exonuclease-deficient replicative DNA pol alpha. Purified exoN has no associated pol activity and catalyzes removal of mispaired nucleotides from DNA duplexes. Consistent with previous reports, it was found that mammalian pol alpha is inefficient at extending from mispaired DNA terminals. However, in similar reactions that included exoN, there was a 4.4-15.7-fold increase in pol alpha-catalyzed elongation from mispaired base pairs. In contrast, exoN did not have a dramatic impact on the ability of exonuclease-deficient variants of Klenow (K-) and T7 polymerase to catalyze extension from mispaired DNA. Continuous DNA replication catalyzed by either pol alpha or K- generated base substitutions at a frequency of 24.3x10(-4) and 38x10(-4), respectively. ExoN restored error-free DNA replication in reactions with pol alpha whereas it did not significantly improve the accuracy of K-. These results are consistent with a functional interaction between exoN and pol alpha to ensure accurate DNA replication.     

Sandhoff disease in Cyprus: population screening by biochemical and DNA analysis indicates a high frequency of carriers in the Maronite community

In the last 15 years, four patients with the infantile form of Sandhoff disease were diagnosed in four different families in Cyprus (population 703,000, birth rate 1.7%). Three of these cases came from the Christian Maronite community (less than 1% of the population) and one from the Greek community (84% of the population). This relatively large number of patients prompted us to initiate an epidemiological study in order to establish the frequency of the mutant allele in Cyprus. Carrier detection was initially based on the measurement of beta-hexosaminidase A and B in both leucocytes and serum. Using the enzyme test, 35 carriers were identified among 244 random Maronite samples and 15 among 28 Maronites with a family history of Sandhoff disease, but only one carrier was found out of 115 random samples from the Greek community. In parallel to the biochemical screening, DNA studies were undertaken in one of the three Maronite patients and in a Greek carrier related to the Greek patient. These studies resulted in the identification of two novel mutations, a deletion of A at nt76 and a G to C transversion at position 5 of the 5'-splice site of intron 8, which have been published. We subsequently screened the carriers detected in the biochemical study for these two mutations using PCR-based tests. Of 50 Maronite carriers examined, 42 were found to have the nt76 deletion. Eight Maronite samples, designated carriers from the biochemical results, were negative for both mutations. It is possible that these individuals were incorrectly classified as carriers since their enzyme values are equivocal, although the presence of another mutation has not been excluded. Two Greek Cypriot carriers and two obligate Lebanese carriers were negative for both mutations. We conclude that there is a high frequency of Sandhoff disease carriers in the Maronite community of Cyprus, approximately 1 in 7, and that a single mutation predominates in this population.     

Delayed reproduction among Great Bitterns Botaurus stellaris breeding in ricefields

Capsule Great Bitterns bred successfully in ricefields in northern Italy, despite a marked delay to the breeding season compared to natural wetlands in the same geographical area.     

Insulinoma--diagnosis and treatment

Insulinomas are the most common functioning endocrine tumors of pancreas. Approximately 10% are multiple, less than 10% can be malignant and 5-10% associated with the MEN-1 syndrome. Insulinomas are the most common cause of hypoglycemia resulting from endogenous hyperinsulinism. The aim of this lecture is to present the up-to-date information concerning the prevalence, diagnosis and treatment of insulinoma.     

Quality of life and marital sexual satisfaction in women with polycystic ovary syndrome

Polycystic ovary syndrome affects 5-10% of women in the developed world, making it the most common endocrine disorder among women of reproductive age. The symptoms typically associated with polycystic ovary syndrome: amenorrhea, oligomenorrhea, hirsutism, obesity, subfertility, anovulation and acne can lead to a significant reduction in female life quality.The aim of the study was to evaluate the effect of polycystic ovary syndrome on quality of life and marital sexual satisfaction. Fifty women with polycystic ovary syndrome were qualified to the study as the research group. The control group consisted of fourty healthy women. A specific questionnaire was used as a research tool in this study. It included the socio-demographic part, polycystic ovary syndrome's symptomatology and validated scales: Polish version of Short Form-36 Health Survey (SF-36) and Index of Sexual Satisfaction (ISS). The mean age of researched women was 28.9+/-5.6 years, and in the control group - 30.5+/-5.3 years (p>0.05). Quality of life parameters for women with polycystic ovary syndrome were lower than for the controls in the aspect of: general health (p<0.01), limitations due to physical health (p<0.05), limitations due to emotional problems (p<0.001), social functioning (p<0.01), energy/fatigue (p<0.001) and emotional wellbeing (p<0.01). Studied women showed worse marital sexual functioning (p<0.05). Marital sexual dysfunctions were diagnosed in 28.6% of women with polycystic ovary syndrome and in 10.5% of healthy women (p<0.05). Polycystic ovary syndrome decreases quality of life and marital sexual functioning among women. A negative effect of hirsutism severity on general well-being and marital sexual life is also observed.     

The effect of resveratrol and its methylthio-derivatives on the Nrf2-ARE pathway in mouse epidermis and HaCaT keratinocytes

Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4′-MTS (S2), 3,5-DM-4′-MTS (S5) and 3,4,5-TM-4′-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH.     

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep‐sea anoxic brine lake as potential polysaccharide degrader

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX‐2^T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B^T from the Shaban deep‐sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant‐derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low‐oxygen conditions. Likewise, proteomics indicated quinone‐mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light‐dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep‐sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase‐rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.