Controlling spontaneous chirality in achiral materials: liquid crystal oligomers and the heliconical twist-bend nematic phase.

Liquid crystal oligomers, namely dimers, trimers and tetramers, consisting of cyanobiphenyl and benzylideneaniline-based mesogenic units connected by either linear or bent alkoxy or alkyl spacers are reported. These materials, although built from achiral molecules, show the spontaneously chiral heliconical twist-bend nematic (NTB) phase. We report the relationships between the shape of the oligomer, and the NTB phase stability, the temperature dependence of the helical pitch length and tilt angle, birefringence, and elastic constants.

Dispersal and immunohistochemical characteristics of neurons in the stem of the porcine vagus nerve.

The present study investigated the distribution and chemical properties of nerve cell bodies within the trunk of the vagus nerve in juvenile female pigs (n=4) using double-labelling immunofluorescence. The neurons appeared mostly as single cells or formed streaks of cells or small ganglia. Many of the perikarya were cholinergic (VAChT-positive; VAChT+) or adrenergic (DßH+) in nature and no SP+ or CGRP+ neurons were encountered. There were no distinct left-right differences regarding the number and chemical coding of the neuronal somata, however, these characteristics significantly varied between particular nerve segments investigated. The vagosympathetic trunks, and thoracic and abdominal segments of the vagus nerve contained on average (the numerical values represent the means for both the left and right corresponding nerve segments) 142, 236, and 111 PGP 9.5-positive neurons, respectively. Proportions of cholinergic and adrenergic neurons were as follows: 0% and 100%, 54.2% and 33.2%, and 52.8% and 35.4%, respectively. Relatively many neurons in the thoracic and abdominal segments stained also for NOS (39.2% and 39.9%, respectively). It remains to be determined whether the porcine intravagal neurons represent a developmental relic, or whether they have any specific functional significance.

Nanomechanical characterization of single phospholipid bilayer in ripple phase with PF-QNM AFM.

Topography and nanomechanical properties of the supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) single bilayer in the ripple phase were investigated by atomic force microscopy with the use of PeakForce Quantitative Nanomechanical Mapping (PF-QNM) mode under liquid conditions. The DMPC single bilayer was deposited on the mica surface by Langmuir-Blodgett and Langmuir-Schaefer techniques combined. Next, the sample was stored overnight at 4 °C under buffer solution (pH = 7.6) in the presence of tris(hydroxymethyl)aminomethane, (Tris), and then, AFM imaging was performed at 21 °C using the same buffer. The AFM topography images revealed the presence of a periodic structure with an average wavelength of 96.0 ± 7.8 nm and an average amplitude of 0.97 ± 0.03 nm. This macro-ripple structure was asymmetric and formed by long ripples composed of two stripes of different heights. The changes in topography were accompanied by local changes in the Young's modulus, which indicate that the nanomechanical properties of the bilayer are not evenly distributed, namely, the higher stripes are characterized by a lower Young's modulus than that calculated for lower stripes of the ripples. This indicates that the ripple phase is composed of phospholipid molecules of periodically changed orientation and conformation characteristic for fluid-like and gel-like state of DMPC.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.).

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein ß2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α7 (Itga7), downregulated in twins, and Nrk, gap junction protein ß6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

Prostaglandin endoperoxide synthase 2 (PTGS2) and prostaglandins F2α and E2 synthases (PGFS and PGES) expression and prostaglandin F2α and E2 secretion following oestrogen and/or progesterone stimulation of the feline endometrium.

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF(2α) synthase (PGFS) and PGE(2) synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E(2) and/or P(4) on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E(2) and/or P(4) treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E(2) plus P(4) at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P(4) at late pseudopregnancy (p < 0.05). Simultaneous incubation with E(2) and P(4) up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E(2) significantly increased PGE(2) secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E(2) and/or P(4) affected neither PGF(2α) secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE(2) secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E(2) and P(4) at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE(2) are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells.

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO2/NO3) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17ß-estradiol (E2; 10⁻9 M) and/or progesterone (P4; 10⁻7 M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻5 M). Prostaglandin F(2α) and PGE2 secretion was measured in medium by ELISA. The pretreatment of cells with P4 (progesterone), E2 (17 ß-estradiol), or E2/P4 augmented TNF-α-induced PGF(2α) and PGE2 secretion (P < 0.01). The pretreatment of cells with E2 or E2/P4 increased NONOate-induced PGF(2α) and PGE2 secretion (P < 0.01). TNF-α induced NO2/NO3 production by BOECs. The pretreatment of cells with E2 augmented only TNF-α-induced NO2/NO3 production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻5 M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

Tumor necrosis factor-α inhibits the stimulatory effect of luteinizing hormone and prostaglandin E(2) on progesterone secretion by the bovine corpus luteum.

Tumor necrosis factor-α (TNF-α) is involved in the tissue remodeling that occurs in the corpus luteum (CL) during its development and regression. This cytokine is also implicated in the regulation of reproduction by its actions on ovarian steroidogenic cells. The aim of this study was to examine the influence of TNF-α on (1) progesterone (P(4)) output by the bovine CL and on (2) the responsiveness of the CL to LH or prostaglandin E(2) (PGE(2)) in vitro. In experiment 1, CL (days 8 to 10 of the estrous cycle) were perfused by using an in vitro microdialysis system with TNF-α (0.1, 0.5, or 1 µg/mL) alone or with TNF-α (1 µg/mL) followed by LH (1000 ng/mL) or PGE(2) (2 × 10(-5) M). Basal P(4) release (P < 0.05) was increased by TNF-α (0.5 or 1 µg/mL). Moreover, TNF-α (1 µg/mL) inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). In experiment 2, 4 h after intrauterine infusion of TNF-α (0.01 µg/mL or 1 µg/mL), CL (days 8 to 10 of the estrous cycle) were collected by colpotomy, cultured, and stimulated with LH (10 ng/mL) or PGE(2) (10(-6) M). Intrauterine infusion of TNF-α at a concentration of 1 µg/mL increased basal P(4) output by CL (P < 0.05). Moreover, the intrauterine infusion of TNF-α at a concentration of 0.01 µg/mL inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). These results indicate that TNF-α (1) does not have an effect on the autonomous, pulsatile release of P(4); (2) increases P(4) secretion by bovine CL with increasing doses, and (3) reduces in a dose-dependent manner the responsiveness of CL to luteotropic factors both directly (after infusion to CL) and indirectly (after intrauterine infusion).

Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium.

The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 microM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS)--proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium--dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.

Intermingled modulatory and neurotoxic effects of thimerosal and mercuric ions on electrophysiological responses to GABA and NMDA in hippocampal neurons.

The organomercurial, thimerosal, is at the center of medical controversy as a suspected factor contributing to neurodevelopmental disorders in children. Many neurotoxic effects of thimerosal have been described, but its interaction with principal excitatory and inhibitory neurotransmiter systems is not known. We examined, using electrophysiological recordings, thimerosal effects on GABA and NMDA-evoked currents in cultured hippocampal neurons. After brief (3 to 10 min) exposure to thimerosal at concentrations up to 100 µM, there was no significant effect on GABA or NMDA-evoked currents. However, following exposure for 60-90 min to 1 or 10 µM thimerosal, there was a significant decrease in NMDA-induced currents (p<0.05) and GABAergic currents (p<0.05). Thimerosal was also neurotoxic, damaging a significant proportion of neurons after 60-90 min exposure; recordings were always conducted in the healthiest looking neurons. Mercuric chloride, at concentrations 1 µM and above, was even more toxic, killing a large proportion of cells after just a few minutes of exposure. Recordings from a few sturdy cells revealed that micromolar mercuric chloride markedly potentiated the GABAergic currents (p<0.05), but reduced NMDA-evoked currents (p<0.05). The results reveal complex interactions of thimerosal and mercuric ions with the GABA(A) and NMDA receptors. Mercuric chloride act rapidly, decreasing electrophysiological responses to NMDA but enhancing responses to GABA, while thimerosal works slowly, reducing both NMDA and GABA responses. The neurotoxic effects of both mercurials are interwoven with their modulatory actions on GABA(A) and NMDA receptors, which most likely involve binding to these macromolecules.

Chromosomal assignment of porcine pregnancy-associated glycoprotein gene family.

This study presents the chromosomal assignment of a multiple pregnancy-associated glycoprotein (PAG) gene family in the domestic pig (pPAG). The pPAG locus was identified by physical mapping (fluorescent in situ hybridisation-FISH; with various probes), and additionally confirmed by Southern hybridisation of pPAG amplicons using laser microdissected Sus scrofa chromosome 1 (SSC1), as genomic templates. Various pPAG probes were produced with the use of diverse identified templates: pPAG1-6, -8, -10 cDNAs (GenBank: L34360-1, AF315377, AF272734, AY188554, AF272735, AY373029 and AY775784, respectively), or genomic DNA (gDNA) probes of pPAG2 gene and its promoter (GenBank: U39198-9, U39762-3, U41421-4). All probes, including long gDNA probes (approximately 9.2kbp GpPAG2 gene; approximately 2.8kbp GpPAG2 promoter), a shorter cDNA probe (PlpPAG4, 1385bp) and amplified pPAG2-like probes (ApPAG2L) specific for cDNA inserts of pPAG2-like gene subfamily (pPAG2, -4, -6, -8 and -10; 1283-1385bp) were produced by random priming using biotin-labelled deoxynucleotides (16-dUTP). Numerous FISH mappings with various pPAG probes revealed the chromosomal assignment of the pPAG gene family to the long arm of porcine chromosome 1 (SSC1q16-q24 region). This cytogenetic assignment was confirmed by Southern hybridisation (with (32)P-labelled pPAG10 probe) of multiple distinct pPAG amplicons (603-3943bp) produced with the use of 25 laser microdissected SSC1, as gDNA templates. This is the first study identifying the chromosomal locus of the pPAG gene family in the pig.

Response to stripe rust (Puccinia striiformis Westend. f. sp. tritici) and its coincidence with leaf rust resistance in hexaploid introgressive triticale lines with Triticum monococcum genes.

Triticale introgressive lines were developed by incorporating diploid wheat (Triticum monococcum [TM16]) genes into the hexaploid triticale genotype LT522/6. The synthetic allotetraploid T. monococcum cereale (A(m) A(m)RR) was used as a bridging form to introduce the genes. A group of 43 introgressive lines, parental stocks and a check cultivar were inoculated at the seedling stage (in the greenhouse) and at the adult plant stage (in the field) with four pathotypes of Puccinia striiformis f. sp. tritici to determine if the stripe rust resistance was derived from TM16 and to analyze the expression of the diploid wheat gene(s) at the hexaploid level. At the seedling stage, 14 triticale introgressive lines expressed resistance to some of the used pathotypes, showing introduction of a genetic material from the T. monococcum genome. Among them, 7 lines were resistant to all four stripe rust pathotypes applied at this stage. In the field, adult plant resistance and percentage of infected leaf area were scored and transformed into the coefficient of infection. Plant response to stripe rust was compatible at these two developmental stages with a high statistical probability showing the genetic dependence on the same genetic background. Also observed was a full concordance of the adult plant resistance to stripe rust with previously assessed resistance to leaf rust, as well as the highly significant linkage of the resistance to the both diseases at the seedling stage in the set of the tested introgression lines. This result strongly suggests that T. monococcum genes responsible for these characters are located in proximity.

Pregnancy-associated glycoprotein family (PAG)--as chorionic signaling ligands for gonadotropin receptors of cyclic animals.

The chorionic pregnancy-associated glycoprotein (PAG) family was identified in pigs, cattle and other eutherian mammals. The objective of this study was to examine whether secretory chorionic proteins (including PAGs), produced in vitro by explants of porcine and bovine placental membranes, may interact with other proteins, i.e. gonadal and extragonadal binding sites. Trophoblast (TRF) and trophectoderm (TRD) explants of pigs (n=38; 14-61 dpc-day post coitum) or cotyledons (CT) of cows (n=5; 40-110 dpc) were long-term cultured. Released chorionic proteins were ultra-fractionated from media (>10 kDa) or precipitated [20-75% of (NH(4))(2)SO(4)]. The PAGs were monitored by Western/PAGE (30-73 kDa). Secretory TRF/TRD/CT (+PAG) proteins (0.78-25 microg/ligand) were examined by radioreceptor assay (RRA) with iodinated hCG ((125)I-hCG) for binding-effectiveness by gonadotropin receptors of cyclic pigs and cows (cRc). Gonadal and extragonadal cRc isolated from luteal-phase corpora lutea and uteri (cCLRc, cMYORc and cENDRc) were tested with positive control ligands: porcine LH and hCG (0.39-50 ng/ml). Control proteins produced in vitro by endometrial (END) explants of cyclic (cEND), pseudopregnant (PsEND) and pregnant (pEND) gilts were utilised as negative ligands (0.78-25 microg/ligand). Positive control ligands competed with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc (18-61%/B(0) for hCG and 27-57%/B(0) for LH). Negative ligands (cEND, PsEND and pEND) did not show cRc bindings. This is the first RRA report indicating that in vitro produced porcine TRF/TRD proteins (+PAG) competed (P< or =0.05) with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc in a concentration- and pregnancy stage-dependent manner. The highest competition with (125)I-hCG (up to P< or =0.001) was found for ultra-fractionated TRF/TRD proteins (>10 kDa) during early pregnancy (<22 dpc). The greatest competition (P< or =0.05) of precipitated porcine TRD proteins (>30 dpc) was detected for fractions obtained by saturation with use of 20% of (NH(4))(2)SO(4). Bovine CT proteins revealed lower competition of (125)I-hCG for bovine cCLRc (during 45 dpc only) that was more efficient with CT (up to 71%) than with non-labelled hCG (82%). The PAG proteins may play a role as potential "signal molecules", because they were able to interact with gonadotropin receptors of luteal-phase animals. It seems that the pPAG proteins may be luteoprotective chorionic-origin signals during implantation and placentation, according to binding-effectiveness of the chorionic ligands that was comparable to LH/hCG ligands with gonadal and extragonadal receptors of cyclic animals.

Radiocompetition of secretory pregnancy-associated glycoproteins as chorionic ligands with luteal and uterine gonadotrophin receptors of pregnant pigs.

Porcine pregnancy-associated glycoprotein (pPAG) family is very promiscuous and its role(s) remains unknown. The objective of this study was to identify whether secretory placental proteins (including pPAGs), produced in vitro by porcine chorionic explants, may interact with other proteins/targets, i.e. luteal and uterine binding sites of pregnant pigs. Trophoblast (TRF) and trophectoderm (TRD) were harvested during peri-implantation and placentation periods (14-61 dpc-day post coitum). In vitro-produced TRF/TRD proteins were isolated from media by ultrafractionation (>10 kDa MWCO) or precipitation with 20-75% saturation of (NH(4))(2)SO(4) and pPAG proteins were monitored by Western blotting. Secretory TRF/TRD ligands (including PAGs) were serially diluted (0.78-25 microg/ligand) and examined by radioreceptor assay (RRA). Luteal and uterine membrane receptors of pregnant pigs (pRc) were isolated from corpora lutea (pCLRc), myometrium (pMYORc) and endometrium (pENDRc). The three pRc types were harvested during three periods of pregnancy: 14 dpc (14 Rc), 21-26 dpc (21-26 Rc) and 31 dpc (31 Rc). The RRA competitions of individual TRF or TRD ligands were performed with (125)I-hCG as tracer and different pRc types. The RRA results of TRF/TRD were compared to hCG/pLH ligands--as positive controls (0.39-50 ng/ml), and endometrial (END) proteins (0.78-25 microg/ml) produced in vitro by END explants of cyclic, pseudopregnant and pregnant gilts (cEND, PsEND and pEND, respectively)--as negative control ligands. Results indicated that secretory TRF/TRD proteins (+pPAGs) were able to compete with (125)I-hCG for binding with other proteins/targets, i.e. luteal and uterine receptors of pregnant pigs (pCLRc, pMYORc and pENDRc) in a concentration- and pregnancy stage-dependent manner. This study indicated that porcine secretory 14-15 dpc TRF (pPAG; 30-73 kDa) ligands, effectively displaced (125)I-hCG tracer from pCL14Rc (up to P< or =0.01), corresponding to displacement by hCG and porcine LH. During the early stage of pregnancy, some competition tendency (P< or =0.01) was also detected for TRF ligands (14-15 dpc) with pEND14Rc. As pregnancy advanced, significant (125)I-hCG competition (at least P< or =0.05) with secretory semi-purified TRD ligands (30-42 dpc) was determined for all types of examined receptors pCL31Rc, pMYO31Rc and pEND31Rc, mainly with TRD fractions precipitated by 20% saturation of (NH(4))(2)SO(4). It seems that chorionic pPAG family can be involved in luteoprotective mechanism during implantation and placentation, according to the binding-interaction with luteal and uterine gonadotropin receptors of pregnant pigs.

Influence of melatonin and its precursor L-tryptophan on Th1 dependent contact hypersensitivity.

There is strong evidence that melatonin possesses immunomodulatory activity. It has been shown that it enhances the immune response, acting as a pro-inflammatory agent. It is suggested that melatonin promotes Th1-mediated immune responses by upregulating IL-12 production by antigen presenting cells. In contrast, it has also been shown that melatonin can inhibit Th1 immunity and instead promote a Th2 response. This discrepancy between different observations of the regulatory activity of melatonin on Th1 immunity encouraged us to further investigate the influence of melatonin and its precursor L-tryptophan on Th1 mediated contact hypersensitivity (CHS). Our results show that both melatonin and L-tryptophan inhibit the inflammatory response associated with CHS. Melatonin inhibited the Th1-dependent immune response by suppressing the production of IFN-gamma and IL-12 by cells in the lymph node. On the other hand treatment with L-tryptophan inhibits CHS without affecting INF-gamma production by Th1 effector cells. Observed suppression of CHS after L-tryptophan treatment is at least partly through the production of the anti-inflammatory cytokine IL-10.

Chorionic mRNA expression and N-glycodiversity of pregnancy-associated glycoprotein family (PAG) of the European bison (Bison bonasus).

Placental PAG mRNA expression and N-glycodiversity of multiple PAG proteins secreted in vitro by trophectoderm (chorion epithelium) of wild pecoran Bovidae taxons was not examined previously. The study on European bison (Eb) aimed: (1) to determine placental PAG mRNA expression by in situ hybridisation; (2) to identify a profile of pecoran PAG protein family secreted in vitro by cotyledonary (CT) explants; (3) to examine N-glycodiversity of the PAG proteins in this wild taxon. In addition, we compared (4) a profile and N-glycodiversity of the PAG protein family secreted in vitro by CT and interCT-trophectoderm (intCT-TRD) explants of domestic ruminants. Cotyledonary sections of the Eb were used for in situ hybridisation (ISH) with (35)S-labelled probes produced with porcine PAG cDNA as templates. Various CT and intCT-TRD explants were long-term cultured in vitro. Chorionic proteins were isolated from media, ultra-filtrated (>10 kDa MWCO) and analysed by PAGE-Western blotting with various polyclonal anti-PAG sera. Protein samples with or without enzymatic deglycosylation were examined after different times of explant cultures. Released chorionic proteins were deglycosylated by N-glycanase F (PNGase F+) and compared to glycosylated forms (PNGase F-). This is the first paper demonstrating the PAG-like mRNA transcript expression (by ISH) and N-glycodiversity of immuno-reactive PAG-like proteins (produced in vitro by chorionic explants) of European bison. Various PAG proteins of Eb (EbPAG) were secreted by CT explants during long-term in vitro studies. Major approximately 78, approximately 67 and approximately 65 kDa EbPAG-like proteins were reduced by enzymatic deglycosylation (at least by 10 kDa). Considerably smaller amounts of approximately 45 kDa EbPAG-like proteins were also observed. In addition, we have found that various PAG proteins (30-73 kDa) were secreted by bovine CT explants, during long-term in vitro cultures. Corresponding amounts of PAG proteins, similar in M(r), were also secreted by intCT-TRD explants, whose tissues were not utilised for PAG protein extraction during other scientists' previous studies. It seems that the M(r)-heterogeneity and N-glycodiversity of the PAG protein family can play very important role during feto-placental interactions in Bovidae species.

DHEAS and POMS measures identify cocaine dependence treatment outcome.

UNLABELLED: Early attrition is a significant problem in the treatment of cocaine dependence, but it is unclear why some patients succeed in treatment while others relapse or drop out of treatment without a demonstrated relapse. The goal of this study was to determine whether baseline levels of select hormones, including the adrenal hormone and excitatory neurosteroid dehydroepiandrosterone sulfate (DHEAS), would distinguish between treatment outcome groups. Based on the literature, completion of 90 days of treatment was established as a key outcome variable. METHODS: Quantitative urine levels of the cocaine metabolite benzoylecgonine (BE) and other substance of abuse analytes, plasma levels of DHEAS, DHEA, cortisol, and prolactin, and the profile of mood states (POMS) were serially measured in 38 male cocaine-dependent (DSM-IV) patients and in 28 controls of similar gender and age over a six month study. Exclusion criteria for the patients and controls included Axis I mood, anxiety or psychotic disorders. The patients could not manifest substance dependence except to cocaine. The patients and controls received remuneration for urine and blood collection. Blood samples for hormone levels were obtained between 8 and 10 a.m. on days 1, 14 and 21 of a 21-day inpatient treatment program and throughout 6 months of outpatient study visits at 45-day intervals. RESULTS: Attrition from treatment and study appointments occurred predominately at the junction between inpatient and outpatient programs. Forty percent of patients made the transition to outpatient treatment and remained abstinent and in treatment for a median of 103 days (ABST). Forty-two percent of patients dropped out of treatment during the inpatient stay or never returned after completing the inpatient program (DO) and 18% had a documented relapse either during, or within the first week after, the inpatient stay (REL). POMS total scores were elevated at treatment entry for both the ABST and DO groups. Plasma DHEAS levels in the DO patients were decreased compared to controls and increased in the ABST patients. POMS total scores for the REL patients at baseline were at control levels. Baseline cortisol levels were not statistically different between the outcome groups, though they were elevated for all cocaine patient groups. When treatment outcome was collapsed into whether patients completed (ABST) or did not complete 90 days of treatment (90N), ABST plasma DHEAS and cortisol were significantly elevated compared to the 90N patients and controls across the first 3 weeks of cocaine withdrawal. CONCLUSIONS: At treatment entry, each of the three patient outcome groups was identified by levels of circulating DHEAS and distressed mood. In the ABST patients, distressed mood during withdrawal may have been mitigated through antidepressant-like actions of enhanced endogenous DHEAS activity, thus contributing to improved abstinence and treatment retention. Patients, such as the DO group, with high levels of distressed mood at treatment entry and low DHEAS levels may benefit from adjunctive pharmacotherapy that targets DHEAS and POMS measures. Patients, such as the REL group, who lack distressed mood at treatment entry, may require intense application of motivational approaches plus residential treatment.

Cocaine addiction as a neurological disorder: implications for treatment.

Clinical and preclinical studies provide convincing evidence for persistent neurological/psychiatric impairments and possible neuronal degeneration associated with chronic cocaine/stimulant abuse. These impairments include multifocal and global cerebral ischemia, cerebral hemorrhages, infarctions, optic neuropathy, cerebral atrophy, cognitive impairments, and mood and movement disorders. These findings may encourage the placement of stimulant addiction into the category of organic brain disorders. Functional and microanatomical anomalies in the frontal and temporal cortex as well as other brain regions may be responsible for certain aspects of phenomenology and neuropsychopathology that are characteristic of stimulant polydrug addictions. These may include broad spectrum of deficits in cognition, motivation, and insight; behavioral disinhibition; attention deficits; emotional instability; impulsiveness; aggressiveness; depression; anhedonia; and persistent movement disorders. Although it is still debated whether the hypofrontality and other brain anomalies observed in stimulant abusers are a consequence or an antecedent of drug abuse, this debate seems purely academic and irrelevant with respect to the importance of compensating for these deficits in the development of treatment strategies. The neuropsychiatric impairments accompanying stimulant abuse may contribute to the very high rate of relapse in addicts that can take place after long periods (years) of abstinence. It is possible that the neurological deficits present in stimulant addicts, whether they are primary or secondary to stimulant abuse, are responsible for perpetual drug abuse which may be a form of self-medication (Weiss et al. 1991, 1992). In this context, addiction to stimulants, once fully developed, may represent a true biological dependency on drugs that temporarily compensate for existing neurological deficits. The concept of self-medication by drug addicts is supported by major theories of biological psychiatry. While a majority of drug addicts are polydrug users, there seems to be a preference for a particular type of drug among different populations of addicts. Addicts who experience distress, anxious dysphoria, and turbulent anger prefer the calming actions of opiates, whereas addicts with preceding attention deficit disorder, depression, or bipolar disorder often prefer stimulants (Khantzian 1985). Figure 1 presents conceptual relationships between brain damage and cocaine/stimulant abuse. More clinical studies are needed to establish unequivocally the epidemiological relationships between preexisting neurological deficits-resulting either from genetic, developmental, traumatic, or neurotoxic factors- and vulnerability to drug addictions. Nonetheless, deducing from the results of preclinical studies, it is conceivable that individuals with neurological deficits associated with attention deficit disorder, developmental neuroanatomical abnormalities, lead poisoning, alcoholism, posttraumatic brain lesions, and PTSD may be more vulnerable to stimulant addiction. This notion has significant empirical support as preclinical studies have shown that animals with lesioned prefrontal cortex became supersensitive to cocaine (Schenk et al. 1991) and animals with lesions at the amygdala, VTA, or raphe nuclei manifest more rapid acquisition of amphetamine self-administration than control rats (Deminiere et al. 1989). The above arguments, postulating neuropathology as an intrinsic component of stimulant addiction, should be taken into consideration with the caveat that the clinical manifestations of the disease are heterogenous and addicts may express varying stages and degrees of the disease as determined by environmental and genetic factors. Therefore, it is likely that stimulant addicts who have less advanced neuropathology may recover spontaneously after detoxification with proper nutritional and psychotherapeutic support if they can sustain abstinence. (ABSTR

Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance.

The abundant CNS cholesterol and its sulfate derivative serve as precursors of different neurosteroids, which bidirectionally modulate neuronal excitability, by potentiating or inhibiting function of the GABAA receptors. The regulation of GABAA receptors in the CNS by the steroids of central or peripheral origin may constitute a vital means of brain-body communication, essential for integrated whole organism responses to external stimuli or internal signals. Modulation of the brain GABA receptors by neurosteroids may form the basis of a myriad of psychophysiological phenomena, such as memory, stress, anxiety, sleep, depression, seizures and others. Therefore, the aberrant synthesis of centrally-active steroids may contribute to defects in neurotransmission, resulting in a variety of neural and affective disorders. The biosynthesis of neurosteroids may also be altered by diet and certain psychotropic drugs, thereby affecting excitation of neurons. Hereditary differences in the level of synthesis and catabolism of different neurosteroids may underlie individual variations in CNS excitability, contributing to differences in personality traits, including the inherited susceptibility to drug addition.