The effect of Artichoke (Cynara scolymus L.) on the expression of calcium-binding proteins in the eggshell gland of laying hens.

The purpose of the present work is to investigate the effect of dietary-supplemented artichoke (Cynara scolymus L.) on the mRNA expression of calbindin 1 (Calb1), osteopontin (Spp1), albumin (Alb) and CALB1 protein in the eggshell gland (ESG) of laying hens. A total of 80 ISA Brown hens (each at 40 weeks of age) were randomly divided into two groups: a control and a treated group. All poultry received 130 g/day of compound feed for laying hens but the treated hens' diet was also supplemented with 3g/kg of dried and milled artichoke (Cynara scolymus L.). The increase of the Ca content in blood of the treated hens was established. Significantly decrease of Spp1 mRNA transcripts was found in the eggshell gland of the treated hens, while the mRNA level of Alb was increased. The relative expression of Calb1 mRNA tended to increase in the treated group. The expression of calbindin protein in the cytoplasm of glandular cells of the shell gland was defined by immunohistochemical method. Very strong signals of calbindin were observed in the treated group. The supplementation of the laying hens' diet with dried artichoke (C. scolymus L.) led to a significant increase of Ca content in blood that was reflected in the changes of expression of the eggshell gland genes involved in the mineralization of eggshell.

Iodine supplementation activates folliculogenesis in rabbit ovary.

The aim of this study was to assess the biological effect of the product Jodis Concentrate (JC) on the rabbit ovaries by evaluating the folliculogenesis and expression of oocyte-specific growth differentiation factor 9 (GDF9).The experiment was conducted with 30 female two month old New Zealand rabbits that were the F1 offspring born to mothers differently treated with Jodis concentrate. The control group (n=10), consisted of F1 offspring born to mothers without iodine treatment, and was not supple- mented with JC. The first experimental group (n=10), consisted of F1 offspring born to mothers treated with JC during pregnancy and the suckling period, and was supplemented with JC daily at a dose of 2 ml/L drinking. The second experimental group (n=10), consisted of F1 offspring born to mothers without iodine treatment, and was also supplemented daily with the same dose of JC - 2 ml/L drinking. All groups were fed with total mixed ration for growing rabbits. The trial lasted 48 days. The ovaries were weighed and prepared for histological examination. The GDF9 protein expression in the ovary was determined by immunohistochemical analysis. The addition of JC to the drinking water of female rabbits led to more active development of the ovarian follicles from primordial to tertiary stage in both experimental groups. More intensive GDF9 protein expression in the oocytes and cumulus cells of rabbits, supplemented with JC was observed.

The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues.

The hepatocyte nuclear factor-4 (HNF-4) contains two transcription activation domains. One domain, activation function-1 (AF-1), consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. Transcriptional analysis of sequential point mutations of the negatively charged residues (Asp and Glu) revealed a stepwise decrease in activity, while mutation of all acidic residues resulted in complete loss of transcriptional activity. Mutations of aromatic and hydrophobic amino acids surrounding the negatively charged residues had a much more profound effect than mutations of acidic amino acids, since even a single mutation of these residues resulted in a dramatic decrease in transactivation, thus demonstrating the importance of hydrophobic residues in AF-1 activity. Like other acidic activators, the AF-1 of HNF-4 binds the transcription factor IIB and the TATA-binding protein directly in vitro. In addition, the cAMP-response-element-binding-protein, a transcriptional adapter involved in the transactivation of a plethora of transcription factors, interacts with the AF-1 of HNF-4 and co-operates in the process of transactivation by HNF-4. The different protein targets of AF-1 suggest that the AF-1 of HNF-4 may be involved in recruiting both general transcription factors and chromatin remodelling proteins during activation of gene expression.

Functional domains of the nuclear receptor hepatocyte nuclear factor 4.

The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128-366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360-366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo- and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.

[Differences in expression and functional organization of the rat tyrosine aminotransferase gene in two lines of Morris hepatoma, 8994 and 7777]. / Razlichiia v ékspressii, funktsional'no'i organizatsii gena tirozinaminotransferazy krys v dvukh liniiakh gepatom Morrisa 8994 i 7777.

Expression and structural organization of tyrosine aminotransferase (TAT) gene in Morris hepatoma cell line 7777 with active and glucocorticoid-inducible TAT gene and in hepatoma 8994, where TAT gene does not function were analysed. No differences in the number of receptor macromolecules, translocation and nuclear binding of hormone-receptor complexes in hormone sensitive (7777) and resistant (8994) cell lines were demonstrated. Dexamethasone increases TAT gene transcription in 7777 cell line but not 8994. Restriction analysis of TAT gene does not reveal any differences either in structural or in regulatory regions. Gel retardation assay with cloned TAT fragment (-400 b.p.) from normal hepatocytes showed identical shift of mobility in 7777 and 8994 cell lines. Moreover, 5'-flanking sequence (-890 b.p.) of TAT gene linked to the bacterial CAT gene is transiently expressed in both cell lines. We have shown that HpaII site (-105 b.p.) of TAT gene is methylated in those cells where TAT gene does not function (thymus, spleen, Zajdela ascites hepatoma) and is demethylated in TAT gene expressing hepatoma 7777 and normal rat hepatocytes. In hepatoma 8994 there are no DNAse I hypersensitive regions, typical to functioning TAT gene from hepatoma 7777 and normal hepatocytes.

In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA.

The skin cells of newborn mice were stably transformed in vivo with the aid of electroporation. The plasmid DNA was introduced subcutaneously followed by high-voltage pulses applied to the skin pleat. NEO-resistant colonies were found in primary cell cultures obtained from the treated skin. The experiments show that in vivo electroporation can be used for the introduction of plasmid DNA into skin cells of mouse.

[Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression]. / Vstraivanie (dA-dT)n-blokov v reguliatornuiu oblast' gena pho5 narushaet ego ékspressiiu.

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.

[Expression of human leukocyte interferon A gene in Saccharomyces cerevisiae under the control of regulatory yeast elements PHO5, GAL1 and GAL10]. / Ekspressiia gena leikotsitarnogo interferona A cheloveka v drozhzhakh Saccharomyces cerevisiae pod kontrolem reguliatornykh élementov drozhzhevykh genov PHO5, GAL1 i GAL10.

A chromosomal gene for human leucocyte interferon A is expressed in Saccharomyces cerevisiae yeasts due to interaction of 5'-nontranslating region of the cloned interferon gene with the regulatory elements of yeast genes PHO5, GAL1 and GAL10. Regulated systhesis of interferon was obtained in all cases. The level of interferon genes expression in case using GAL1 and GAL10 genes regulatory elements (5 X 10(5) and 5 X 10(6) u X l-1) correlated with the distances to their promoters. The highest yield of interferon (10(8) u X l-1) was obtained when the PHO5 gene regulatory elements were used.

[Age and changes in the hematocrit in the mesenteric capillaries of the white rat]. / Vozrastnye izmeneniia pokazatelia gematokrita v kapilliarakh bryzheiki beloi krysy.

The dynamics of the capillary hematocrit in 4-5-, 7- and 11-week rats involved its reduction with ageing from 0.17 l/l in 4-5-week rats to 0.10 l/l in 7- and 11-week ones in spite of considerable variability in each animal group. The reduction of the hematocrit seems to be due to the growth of capillary net in mesentery.

[Development of the reactivity of the microcirculatory system of the rat mesentery during sexual maturation]. / Stanovlenie reaktivnosti mikrotsirkuliatornoi sistemy bryzheiki krysy v period polovogo sozrevaniia.

Biomicroscopy was employed to study the time-course of microcirculatory reactivity in rat mesentery under the influence of adrenaline solution from the 3d to the 13th week of development. Variation in the microcirculatory reactivity in the mesentery in ontogenesis is associated with an increase in the sensitivity of microvessels to the action of vasoactive substances and with the organization of the microcirculatory bed becoming more complicated. Three stages were revealed in the development of microvascular reactivity in the course of formation of mesenterial microcirculation: early (3-5 weeks), characterized by relatively low sensitivity of microvessels; intermediate (7-8 weeks) marked by a progressive increase in microvascular sensitivity; mature (starting from the 13th week) characterized by the appearance of the definitive reaction pattern.

[Structural changes and hemodynamic relations in the microcirculatory bed of the mesentery of the white rat during the period of sexual maturation]. / Strukturnye izmeneniia i gemodinamicheskie otnosheniia v mikrotsirkuliatornom rusle bryzheiki beloi krysy v period polovogo sozrevaniia.

Structural changes and hemodynamic relations have been studied in the microcirculatory bed of the white rat small intestine mesentery during sex maturation (from the 3d up to the 10th week of the postnatal development). All calculations are performed regarding the mesenteric segment limited with two intestinal arteries, which is considered as an elementary microvascular module. Complication of the microcirculatory bed construction takes place at the expense of increasing number (nearly five-fold) of microvessels in the segment and increase of the capillary network density. The hemodynamic factor plays a certain role for stimulating the process of the capillary growth. The definitive structure of the mesenteric microcirculatory bed is completed by the 7th week. The main rearrangement of the microcirculatory system during the developmental process from a simple arterio-venular loop up to a complex microcirculatory bed with a branching capillary network is performed within the limits of the mesenteric segment.

[Erythrocyte concentration in vessels of the microcirculatory bed of the mesentery of the white rat (according to the results of in vivo microcinematography)]. / Kontsentratsiia éritrotsitov v sosudakh mikrotsirkuliatornogo rusla bryzheiki beloi krysy (po dannym prizhiznennoi mikrokinos"emki).

The red cells velocity and hematocrit value were studied with high-speed microfilming in microvessels of albino rat mesentery. Decrease of the red cells velocity and increase in the number of microvascular branching were followed by a decrease in the hematocrit value: 20% in the precapillary arterioles and 16% in capillaries. The concentration of erythrocytes increased to 26% in venules. The magistral communication between arterioles and venules has a specific place among microvessels. These channels are relatively wider, the red cells velocity is high (0.9 mm/sec) as well as the hematocrit value (23%). Variation of hematocrit value in capillaries is not solely the function of their volume but also the function of their position in capillary net.