Evaluation of reference genes for quantitative real-time PCR in Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction and differentiation.

Quantitative real time reverse transcription PCR, qRT-PCR, is one of the most important techniques for assessing the level of gene expression. Selecting the correct reference gene to normalize the results is a key step in this method. Inaccurate data can be generated if the correct reference gene is not selected. The level of the expression of reference genes is tissue-variable, and in the case of mesenchymal stem cells (MSC), it can be different depending on the source of their origin. The aim of this study was to select the reference gene for Wharton's Jelly-derived MSC (WJ- MSC) that were undergoing transduction and differentiation. In this work, the expression of 32 genes was analyzed, of which two (RPS17 and 18S rRNA), which had the most stable expression level, were selected. A comparative analysis of the expression stability of the selected genes was then performed with the genes that are most commonly used in the literature, i.e. ß-actin and GAPDH. Next, it was determined that a false picture of the expression level of the studied genes can be obtained when a reference gene with variable expression level is used for normalization. RPS17 and 18S rRNA proved to be the most stable reference genes for the WJ-MSC that had been subjected to the lentiviral transfection procedure followed by differentiation. The expression of ß-actin and GAPDH was highly unstable and therefore these genes are not suitable for use as reference genes in studies involving WJ- MSC.

Primary aldosteronism in pregnancy.

We describe a case of primary aldosteronism secondary to bilateral adrenal hyperplasia in three subsequent pregnancies. The disease was diagnosed soon after the first pregnancy, which ended in a miscarriage, and was treated pharmacologically with spironolactone. Because spironolactone is contraindicated in pregnancy, while the hypotensive effects of methyldopa, hydralazine, labetalol, diazoxide and nifedipine were unsatisfactory, in the second (since week 14) and third (since week 6) pregnancy, she received amiloride. This agent, administered at a daily dose of 10-15 mg, effectively controlled blood pressure, reversed hypokalaemia, and led to an increase in plasma renin activity. The course of both pregnancies was uneventful and ultrasonography performed during each visit revealed normal foetal development without growth retardation. Both pregnancies ended in giving birth to healthy children. Our report shows that each case of treatment-resistant hypertension in pregnancy requires assessment for the presence of primary aldosteronism, and that amiloride seems to be a safe and effective agent in the non-surgical treatment of this disorder in pregnant women with primary aldosteronism.

Transcriptional activity of telomerase complex in CD34- stem cells of cord blood in dependence of preparation time.

The aim of the study was to determine whether the expression of telomerase subunits encoding genes changes during the process of cord blood preparation. It should establish if the commonly accepted 24 hours time interval in stem cells kriopreservation procedure significantly influences their immortalization and so decreases the "quality" of cord blood stem cells. Investigation includes 69 women. Spontaneous labour was the inclusion condition. The material was collected at birth after clamping of umbilical cord by direct vasopuncture. CD34- cells were extracted from cord blood (MACS, Miltenyi Biotec; Bisley, Surrey, UK). The expression profile of telomerase activators and inhibitors encoding genes was determined using HG_U133A oligonucleotide microarray (Affymetrix). We used a real-time quantitative RT-PCR assay to quantify the telomerase TERT, hTR and TP1 subunits mRNA copy numbers in CD34- cells in 0, 6, 12 and 24 hours after cord blood collection. We observed significant decrease of numbers of copies of TERTA+B mRNA within the successive hours of observation. Significant decrease of numbers of TERTA mRNA copies was confirmed after 24 hours. However, we observed significant increase of numbers of copies of TERTB mRNA after 6 hours of observation. Similar level was maintained during another 6h. The significantly lower number of copies of TERTB mRNA was observed after 24h. We also observed significant increase of number of copies of TERT mRNA after 6 hours. Number of copies of TERT mRNA significantly decreased after another 6h, remaining, however, on a higher then initial one. The significant lower number of copies of TERT mRNA was observed 24h after delivery. The possible explanation of those results is discussed in the paper.

[The "Bizzare" leiomyoma in histopathological examination of the uterine myoma: dilemma of the range of operative treatment]. / "Bizzare" leiomyoma w badaniu histopatologicznym miesniaka trzonu macicy--dylemat zakresu leczenia operacyjnego.

The very rare case of leiomyoma sarcomatosum was presented and differentiated to "bizzare" leiomyoma at 39 years old woman, who was conservative preoperation diagnosis of uterine myoma. To get the after-operation prognosis the following elements of tumour have been studied: mitotic index, presence and type of necrosis, separation of the tumour and perivascular infiltration. A decision of conservative treatment was confirmed by shown world wide literature.

Influence of L-naphthylalanine in position 3 of AVP and its analogues on their pharmacological properties.

We describe the synthesis and pharmacological properties of two series of analogues: one which consists of three peptides having L-1-naphthylalanine in position 3 and the second composed of analogues substituted in position 3 with L-2-naphthylalanine. All peptides were tested in bioassays for pressor and antidiuretic activities. We also checked the uterotonic activity in vitro. We observed that the activity of counterparts in both series is, in two cases, strikingly different. One of the new analogues, [(L-2-Nal)3,(D-Arg)8]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first potent V1 antagonist devoid of antiuterotonic activity. This analogue was designed without modification of position 1, which was previously thought to be essential for substantial pressor antagonism. Two other peptides, [Mpa1;(L-2-Nal)3;(D-Arg)8]VP and [Mpa1,(L-1-Nal)3,D-Arg)8]VP, are highly potent V2 agonists. The second analogue is highly selective. With the exception of [(L-2-Nal)3]AVP, which showed weak antioxytocic activity, (L-Nal)3 modification resulted in the almost complete removal of interaction of our analogues with oxytocic receptors in vitro. Our results suggest that position 3 in AVP and its analogues is important not only for binding and recognition as previously though, but also for pressor, antidiuretic and uterotonic activities. We also assume that the hindering effect caused by bulky naphthyl moiety has a significant impact on the bioactive conformations of molecules which contain Nal residue, and can thus influence their interaction with V1, V2 and oxytocic receptors.

Surprising pharmacological activity of analogues designed by substitution of position 3 in arginine vasopressin (AVP) and 8-D-arginine vasopressin with L-2-napthylalanine.

In an attempt to develop more active and selective analogues of arginine vasopressin (AVP), two peptides have been designed, synthesized and tested for vasopressor (V1-receptors) and antidiuretic (V2-receptors) activities. We also estimated the uterotonic and anti-uterotonic activities of these compounds in-vitro. The first peptide, [(L-2-Nal)3] AVP is a highly active V2-agonist. The second analogue, [(L-2-Nal)3, (D-Arg)R]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first V1-antagonist devoid of anti-uterotonic activity. High antipressor potency of the second peptide was achieved without modification of position 1.