Genistein and daidzein affect in vitro steroidogenesis but not gene expression of steroidogenic enzymes in adrenals of pigs.

Phytoestrogens (PEs), including genistein and daidzein, are plant-derived substances that mimic or antagonize estrogen action in animals. The majority of studies investigated the effects of PEs on reproduction in humans and laboratory animals. The mechanisms of phytoestrogen action on reproductive processes in domesticated animals, including pigs, are garnering increasing attention. However, very few in vivo and in vitro studies investigating the effects of PEs on adrenal glands have been carried out on models other than humans and rats. The aim of the present study was to determine whether the effects of genistein and daidzein on adrenal in vitro steroidogenesis are accompanied by changes in expression of genes encoding key steroidogenic enzymes in porcine adrenocortical cells. The following genes were analyzed: cholesterol side-chain cleavage enzyme (P450scc, CYP11A1 gene), 3ß-hydroxysteroid dehydrogenase (3ß-HSD, HSD3B1 gene), 17α-hydroxylase/C17-20 lyase (P450c17, CYP17A1 gene) and 21-hydroxylase (P450c21, CYP21A2 gene). Porcine adrenocortical cells collected from both luteal- and follicular-phase gilts were exposed for eight hours to genistein (10 µM), or daidzein (10 µM), in the absence or presence of ACTH (5 nM). Genistein and daidzein inhibited basal and ACTH-stimulated secretion of cortisol and corticosterone and stimulated secretion of androstenedione. PEs did not affect the expression of CYP11A1, HSD3B1, CYP17A1 and CYP21A2 in the adrenocortical cells of luteal- and follicular-phase gilts. It can be concluded that the influence of PEs on steroid secretion in porcine adrenal glands is not mediated by changes in the expression of genes encoding major steroidogenic enzymes. More studies are needed to elucidate the intracellular mechanisms leading to the PE-induced changes in adrenal steroidogenesis in pigs.

Expression of adiponectin and its receptors in the porcine hypothalamus during the oestrous cycle.

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2-3, 10-12, 14-16 and 17-19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14-16, whereas in SME, the most pronounced gene expression was found on days 2-3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17-19 and in POA on days 2-3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14-16. AdipoR1 gene expression in POA was potentiated on days 2-3 and 10-12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2-3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2-14 of the cycle), and they decreased on days 17-19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2-3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2-3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2-3 (p < 0.05), while in POA on days 17-19 and in SME on days 10-12 and 14-16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary.

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP - on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) - on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.

Hemodynamic modeling of the intrarenal circulation.

Three dimensional, time dependent numerical simulations of healthy and pathological conditions in a model kidney were performed. Blood flow in a kidney is not commonly investigated by computational approach, in contrast for example, to the flow in a heart. The flow in a kidney is characterized by relatively small Reynolds number (100 < Re < 0.01-laminar regime). The presented results give insight into the structure of such flow, which is hard to measure in vivo. The simulations have suggested that venous thrombosis is more likely than arterial thrombosis-higher shear rate observed. The obtained maximum velocity, as a result of the simulations, agrees with the observed in vivo measurements. The time dependent simulations show separation regimes present in the vicinity of the maximum pressure value. The pathological constriction introduced to the arterial geometry leads to the changes in separation structures. The constriction of a single vessel affects flow in the whole kidney. Pathology results in different flow rate values in healthy and affected branches, as well as, different pulsate cycle characteristic for the whole system.

Identification of chosen apoptotic (TIAR and TIA-1) markers expression in thyroid tissues from adolescents with immune and non-immune thyroid diseases.

The aim of this study was to estimate sodium iodide symporter (NIS) and thyroid peroxidase (TPO) expression in thyrocytes from patients with GD and no-toxic multinodular goitre (NTMG) in relationship with apoptotic (TIAR and TIA-1) markers. The investigation was performed on thyroid cells isolated from postoperation thyroid tissues from 15 patients aged 12-21 years old with GD and 15 cases aged 13-21 years old with NTMG. Detection of NIS and TPO was performed by immunohistochemistry. Analysis of apoptotic markers in thyroid tissues was performed using antibodies to TIAR and TIA-1 by Western Blot and immunohistochemistry. Identification of proapoptotic TIAR and TIA-1 molecules in the thyroid tissues revealed a higher expression of both proteins in patients with Graves' disease (+++; +, respectively) in comparison to patients with NTNG (+; 0). In addition, TIAR expression was detected in three bands [p50, p42, p38 (kDa)] and TIA-1 in two bands [p22, p17 (kDa)]. using Western Blot test in patients with thyroid autoimmune diseases. In patients with NTNG expression of both apoptotic proteins was lower and identified in single bands: 42 (kDa) for TIAR and 17 (kDa) for TIA-1. The analysis of expression of NIS and TPO in thyroid follicular cells was higher in patients with Graves' disease in compared to their detection in patients with NTMG. In addition, degree of thyroid antigen expression positive correlated with amount of proapoptotic markers (TIAR, p<0.001; TIA-1, p<0.025 for NIS; TIAR, p<0.012 for TPO). We conclude that elevated expression of NIS and TPO in Graves' disease is associated with higher stimulation and activation of apoptosis in thyroid follicular cells during autoimmune process.

A comparison of coding sequence and cytogenetic localization of the myostatin gene in the dog, red fox, arctic fox and Chinese raccoon dog.

The gene encoding myostatin (MSTN), due to its crucial function for growth of skeletal muscle mass, is an important candidate for muscularity. In this study we analyzed the nucleotide sequence and FISH localization of this gene in 4 canids, including 3 farm species. The nucleotide sequence of the MSTN coding fragment turned out to be highly conserved, since its identity among the studied species was very high and varied between 99.4 and 99.7%. Only 1, widely spread, silent single nucleotide polymorphism (SNP) was found in exon 1 of the Chinese raccoon dog. The MSTN gene was localized close to the centromere in one-armed chromosomes of the dog (37q11) and bi-armed chromosomes of the red fox (16p11) and arctic fox (10q11), with an exception of the Chinese raccoon dog chromosome (2q14-q21). This chromosome is orthologous to 3 canine chromosomes and thus the MSTN was found more interstitially. Our results are in agreement with the hypothesis that karyotypes of the canids evolved mainly through centric fusion/fission events, while tandem fusions occurred rarely.

Impact of inlet channel geometry on microfluidic drop formation.

We study the impact of inlet channel geometry on microfluidic drop formation. We show that drop makers with T-junction style inlets form monodisperse emulsions at low and moderate capillary numbers and those with Flow-Focus style inlets do so at moderate and high capillary numbers. At low and moderate capillary number, drop formation is dominated by interfacial forces and mediated by the confinement of the microchannels; drop size as a function of flow-rate ratio follows a simple functional form based on a blocking-squeezing mechanism. We summarize the stability of the drop makers with different inlet channel geometry in the form of a phase diagram as a function of capillary number and flow-rate ratio.

[Organization of the Helicobacter pylori genome]. / Organizacja genomu Helicobacter pylori.

Helicobacter pylori is a Gram-negative, spiral-shaped pathogenic bacterium that was firstly isolated and cultured from biopsy specimens by Marshall and Warren in 1983. This organism is a human gastric pathogen associated with peptic ulcer disease as well as chronic gastritis. Recent epidemiological studies have demonstrated that H. pylori is a primary risk factor for the development of intestinal type gastric adenocarcinoma. H. pylori is the first bacterium for which the genomes of two unrelated strains (26695 and J99) have been sequenced. The genome of H. pylori is relatively low in size (1.6-1.73 Mb). In this review, we compare the organization of two sequenced H. pylori genomes. A special emphasis on genetic diversity of H. pylori including plasticity zone and cag pathogenicity island has been placed.

Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA.

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Sequence recognition, cooperative interaction, and dimerization of the initiator protein DnaA of Streptomyces.

Using a combined PCR-gel retardation assay, the preferred recognition sequence of the Streptomyces initiator protein DnaA was determined. The protein showed a preference toward DNA containing two Escherichia coli-like DnaA boxes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas the consensus sequence of the DnaA boxes found in the Streptomyces oriC region is TTGTCCACA). In quantitative band shift experiments, the kinetics of the Streptomyces DnaA-DnaA box interaction was characterized. The DnaA protein can form dimers while binding to a single DnaA box; dimer formation is mediated by the domain III of the protein, and the dissociation constant of this process was between 35 and 115 nm. Streptomyces initiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance separating the DnaA boxes, domain I (or I and II) is responsible. The cooperativity constant is moderate and is in the range of 20-110.

Sequence-selectivity of 5,11-dimethyl-5H-indolo[2,3-b]quinoline binding to DNA. Footprinting and molecular modeling studies.

Indolo[2,3-b]quinolines are a new family of the DNA intercalators showing significant cytotoxic activity. The mechanism of their action is based on the inhibition of DNA topoisomerase II activity. It depends on their ability to induce and stabilize drug-topII-DNA cleavable complexes. Site-specific intercalation of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ) was analyzed in vitro by DNaseI footprinting and by molecular modeling. To model the DNA-intercalator complex, use was made of the CVFF and ESFF force fields implemented in Insight 97.0 software. Experimental results were verified using a simple statistical model. The DiMIQ molecule was found to bind preferentially to the pBR322 DNA plasmid in the 5'-TGCTAACGC-3' region between adjacent adenine bases.

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes.

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.

Structure and regulation of the dnaA promoter region in three Streptomyces species.

The regulatory region of the Streptomyces dnaA gene comprises a single promoter and two DnaA boxes that are located upstream of the promoter. Comparative analysis of the dnaA promoter region from S. chrysomallus, S. lividans and S. reticuli revealed that the location, spacing and orientation of the DnaA boxes are conserved. In vitro studies demonstrated that efficient binding of the Streptomyces DnaA protein to DNA requires the presence of two DnaA boxes. In vivo analysis of dnaA promoter mutants deleted for one or both DnaA boxes indicated that the dnaA gene is autoregulated. However, the degree of derepression observed is relatively modest.

Functional domains of DnaA proteins.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Interactions of the Streptomyces lividans initiator protein DnaA with its target.

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Rare, suppurative pulmonary infection caused by Nocardiopsis dassonvillei recognized by glycolipid markers.

An opportunistic actinomycete was isolated as the only etiological agent of a severe, suppurative pulmonary infection. The strain was rapidly recognised as Nocardiopsis by the taxonomically important and immunologically active glycolipid markers (G1 and G2). Identification of the clinical isolate, from a group of actinomycetes mainly known as soil habitants, was definitely proved by chemotaxonomic studies (cell wall/sugar, phospholipid and fatty acid types) as well as by genomic data (GC content, DNA-DNA reassociation). The level of DNA-DNA homology of the clinical actinomycete, in comparison with other reference members of this genus, revealed the highest (88%) relatedness to Nocardiopsis dassonvillei. The results confirmed the value and generic specificity of glycolipid markers from Nocardiopsis, the first time used for rapid recognition of a clinical strain causing a nocardiosis-like disease.