Chorionic expression of heterogeneous products of the PAG (Pregnancy-Associated Glycoprotein) gene family secreted in vitro throughout embryonic and foetal development in the pig

Porcine PAG (pPAG) are placental products of a multigene family that is strongly expressed in the chorionic epithelium (trophoblast and trophectoderm). The objective of this study was to define a pattern of the pPAG proteins, secreted in vitro by chorionic explants harvested on 16-77 days of pregnancy. Trophoblastic and trophectodermal explants were collected from pregnant (PR) gilts (n = 27) and used for protein in vitro production (8-261 h). Endometrial explants of luteal-phase gilts (E10, n = 4) and pseudopregnant gilts (PsE, n = 2) were used as negative controls for protein immunoblotting. Proteins (PR, E10, PsE) were isolated mainly from incubation media, fractionated, dialysed and separated by SDS-PAGE. Heterogeneous Western blotting with various polyclonal anti-PAG sera raised against bovine or ovine antigens (anti-bPAG, or anti-oPAG) initially identified the pPAG proteins. Such blotting of fractionated chorionic proteins allowed for the isolation of porcine antigens that were employed as immunogens to raise several homologous antisera (anti-pPAG). Crude antisera were adsorbed on endometrial extracts or proteins of non-PR pigs, to remove non-relevant antibodies. The patterns of pPAG proteins secreted in vitro varied throughout pregnancy (35-72 kDa). During implantation, approximately 43 kDa (Day 16) or approximately 68.1 kDa (Days 17-25) pPAG proteins were detected. During placentation and as pregnancy advanced (Days 31-77), approximately 72.3 kDa pPAG proteins were observed. The secretions of parallel multiple smaller proteins (35.4-47.2 kDa), presumably, as forms of processed pPAG precursors, increased with the progress of gestation. In conclusion, the pPAG protein family plays a very important role during implantation, placenta formation and embryonic/foetal development in the pig.     

Akt signaling regulates side population cell phenotype via Bcrp1 translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.     

Psychological stress exerts an adjuvant effect on skin dendritic cell functions in vivo

Psychological stress affects the pathophysiology of infectious, inflammatory, and autoimmune diseases. However, the mechanisms by which stress could modulate immune responses in vivo are poorly understood. In this study, we report that application of a psychological stress before immunization exerts an adjuvant effect on dendritic cell (DC), resulting in increased primary and memory Ag-specific T cell immune responses. Acute stress dramatically enhanced the skin delayed-type hypersensitivity reaction to haptens, which is mediated by CD8(+) CTLs. This effect was due to increased migration of skin DCs, resulting in augmented CD8(+) T cell priming in draining lymph nodes and enhanced recruitment of CD8(+) T cell effectors in the skin upon challenge. This adjuvant effect of stress was mediated by norepinephrine (NE), but not corticosteroids, as demonstrated by normalization of the skin delayed-type hypersensitivity reaction and DC migratory properties following selective depletion of NE. These results suggest that release of NE by sympathetic nerve termini during a psychological stress exerts an adjuvant effect on DC by promoting enhanced migration to lymph nodes, resulting in increased Ag-specific T cell responses. Our findings may open new ways in the treatment of inflammatory diseases, e.g., psoriasis, allergic contact dermatitis, and atopic dermatitis.     

The effect of leukaemia inhibitory factor on SOD1 G93A murine amyotrophic lateral sclerosis

Before potential therapeutic strategies for the treatment of amyotrophic lateral sclerosis (ALS) can be advanced to human clinical trials, there is a need to assess them in an animal model that best resembles the disease process. SOD1 G93A mice have close resemblance to familial ALS (fALS) and have been used in this study to evaluate the therapeutic potential of leukaemia inhibitory factor (LIF). LIF action was investigated by assessing three delivery methods: (1) daily subcutaneous injection; (2) through LIF rods placed adjacent to hind limb skeletal muscle and (3) continuous intrathecal infusion. The effect on disease progression was assessed by semi-quantitative and quantitative functional measurements, and histologically on the survival of motor neurons and number of reactive astrocytes. The results show that LIF had no beneficial effects when administered using the three methods of drug delivery. These results suggest that further evaluation of LIF in this transgenic model is required to fully characterize its' therapeutic potential.     

Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjectedto 15 d of nitrogen deprivation were resupplied for 10 d with1.0 mol m^–3 nitrogen provided as NO₃^–, NH₄⁺, orNH₄⁺+NO₃^– in flowing hydroponic culture. Plants in a fourthhydroponic system received 1.0 mol m^–3 NO₃^– duringboth stress and resupply periods. Concentrations of solublecarbohydrates and organic acids in roots increased 210 and 370%,respectively, during stress. For the first day of resupply,however, specific uptake rates of nitrogen, determined by ionchromatography as depletion from solution, were lower for stressedthan for non-stressed plants by 43% for NO₃^- resupply, by 32%for NH₄⁺ + NO₃^– resupply, and 86% for NH₄⁺ resupply. Whenspecific uptake of nitrogen for stressed plants recovered torates for non-stressed plants at 6 to 8 d after nitrogen resupply,carbohydrates and organic acids in their roots had declinedto concentrations lower than those of non-stressed plants. Recoveryof nitrogen uptake capacity of roots thus does not appear tobe regulated simply by the content of soluble carbon compoundswithin roots. Solution concentrations of NH₄⁺ and NO₃^– were monitoredat 62.5 min intervals during the first 3 d of resupply. Intermittent‘hourly’ intervals of net influx and net effluxoccurred. Rates of uptake during influx intervals were greaterfor the NH₄⁺ -resupplied than for the NO₃^– -resuppliedplants. For NH₄⁺ -resupplied plants, however, the hourly intervalsof efflux were more numerous than for NO₃^– -resuppliedplants. It thus is possible that, instead of repressing NH₄⁺influx, increased accumulation of amino acids and NH₄⁺ in NH₄⁺-resupplled plants inhibited net uptake by stimulation of effluxof NH₄⁺ absorbed in excess of availability of carbon skeletonsfor assimilation. Entry of NH₄⁺ into root cytoplasm appearedto be less restricted than translocation of amino acids fromthe cytoplasm into the xylem. Key words: Ammonium, nitrate, nitrogen-nutrition, nitrogen-stress, soybean     

Sequencing of the ovine interleukin-8-encoding cDNA using the polymerase chain reaction

The nucleotide (nt) sequence encoding the ovine homologue of interleukin-8 (IL-8) was determined. The mRNA is 1494-nt long with an ORF of 101 codons. The long 3' non-coding element contains several ATTTA repeats implicated in the swift turnover of other chemokine mRNAs. The encoded protein of 11 kDa before processing, and 9 kDa as mature protein, contains the Cys-Xaa-Cys motif common to -chemokines, and has conserved amino acids (aa) at positions identified as receptor contact sites for IL-8. Identities with other published IL-8 aa sequences are: dog, 91%; pig, 87%; rabbit, 84%; human, 78%; guinea pig, 69%. A 49% aa identity is also found with a chicken embryo fibroblast protein.     

Further Characterization of the Long-Term Effect of RU24722 on Tyrosine Hydroxylase in the Rat Locus Coeruleus

Abstract: Recent data have indicated that the long-lasting increase in tyrosine hydroxylase (TH) protein could be differently expressed in the anterior and posterior locus coeruleus (LC) after a single intraperitoneal injection of RU24722, which has been proposed as a potent activator of catecholaminergic systems. In the present study, we have evaluated the dose and time course responses and the effect of a repeated treatment with RU24722 at 3-day intervals on TH protein level in the anterior and posterior rat LC. The results showed that RU24722 induces a long-lasting increase of TH protein level in the anterior and posterior LC that was maximal 3 days following a single injection of 30 mg/kg. The increase in TH protein was maintained at a constant level after repeated administrations of RU24722 at 3-day intervals. Furthermore, we have investigated whether the effect of the drug on TH protein could be modulated via several hormonal systems. The long-term increase of TH steady-state content after RU24722 was still observed 15 days after castration, adrenalectomy, hypophysectomy, and thyroidectomy. The initial steady-state TH protein level was significantly higher in the anterior LC of thyroid- or hypophysectomized and in the posterior LC of hypophysectomized rats. However, this increase was reversed when animals were housed at 28°C.