Solid phase DNA amplification: a simple Monte Carlo Lattice model

Recently, a new way to amplify DNA, called solid phase amplification (SPA), has been introduced. SPA differs from the traditional polymerase chain reaction (PCR) in the use of surface-bound instead of freely-diffusing primers to amplify DNA. This limits the amplification to two-dimensional surfaces and therefore allows the easy parallelization of DNA amplification in a single system. Furthermore, SPA could provide an alternate route to DNA target implantation on DNA chips for genomic studies. Standard PCR processes are usually characterized (at least initially) by an exponential growth and a broad population distribution, and they are well described by the theory of branching processes, wherein a generating function can be used to obtain the probability distribution function for the population of offspring. This theoretical approach is not appropriate for SPA because it cannot properly take into account the many-body (steric) and geometric effects in a quenched two-dimensional environment. In this article, we propose a simple Lattice Monte Carlo technique to model SPA. We study the growth, stability, and morphology of isolated DNA colonies under various conditions. Our results indicate that, in most cases, SPA is characterized by a geometric growth and a rather sharp size distribution. Various non-ideal effects are studied, and we demonstrate that such effects do not generally change the nature of the process, except in extreme cases.     

Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efficiently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Interestingly, UGT2B10 and UGT2B11, which are considered as orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, conjugated 12-HETE, 15-HETE, and 13-HODE. In summary, our data showed that several members of UGT1A and UGT2B families are capable of converting LA and AA metabolites into glucuronide derivatives, which is considered an irreversible step to inactivation and elimination of endogenous substances from the body.     

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.     

Long-chain alkenones and related compounds in the benthic haptophyte Chrysotila lamellosa Anand HAP 17

The neutral lipid compositions of the coastal haptophyte Chrysotila lamellosa HAP 17 grown in batch culture at 10 and 20 degrees C have been determined. A comparison was also made between the lipid compositions of cells harvested in early and late stationary phase. This species contains a suite of very long-chain C(37)-C(40) alkenones and alkenoates as found in a few microalgae from the Haptophyta. The distributions of these compounds show some differences to earlier reports of different strains of this alga, which are only in part attributable to culture conditions. A suite of long-chain alkenols, the reduced form of the alkenones, was characterized for the first time. The abundance of these compounds was only 1.5% of that of the corresponding alkenones, and the relative proportion of C(37)-C(38) constituents depended on growth temperature. These data show that haptophyte algae are a possible source of the alkenols found in some marine sediments, but the small amounts found suggest that other sources such as bacterial reduction of alkenones are more likely in highly reducing sediments. A mixture of C(29)-C(33) n-alkenes, dominated by the C(31:1) monoene, was found in marked contrast to previous analyses of other strains which reported only the presence of a C(31:2) diene. The sterol distribution included the common haptophyte sterol 24alpha-methylcholesta-5,22E-dien-3beta-ol (epi-brassicasterol) as well as significant amounts of Delta(5)- and Delta(5,22)-C(29) sterols.     

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.