Optimization of horse chestnut (<Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.) somatic embryo conversion

Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol) in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets (43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of converted plantlets was assessed by flow cytometry and no major ploidy changes were found.     

New progesterone receptor antagonists: phosphorus-containing 11beta-aryl-substituted steroids

A new series of phosphorus-containing 11beta-aryl-substituted steroids have been synthesized in an eight-step sequence involving a palladium-catalyzed coupling reaction to introduce a phosphorus group onto the aromatic ring. The compounds were evaluated for progesterone receptor (PR) antagonist activity in a T47D cell-based assay and for glucocorticoid receptor (GR) antagonist activity in an A549 cell-based assay. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in vivo in a rat complement C3 assay.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.     

The philosophical "mind-body problem" and its relevance for the relationship between psychiatry and the neurosciences

Parallel to psychiatry, "philosophy of mind" investigates the relationship between mind (mental domain) and body/brain (physical domain). Unlike older forms of philosophy of mind, contemporary analytical philosophy is not exclusively based on introspection and conceptual analysis, but also draws upon the empirical methods and findings of the sciences. This article outlines the conceptual framework of the "mind-body problem" as formulated in contemporary analytical philosophy and argues that this philosophical debate has potentially far-reaching implications for psychiatry as a clinical-scientific discipline, especially for its own autonomy and its relationship to neurology/neuroscience. This point is illustrated by a conceptual analysis of the five principles formulated in Kandel's 1998 article "A New Intellectual Framework for Psychiatry." Kandel's position in the philosophical mind-body debate is ambiguous, ranging from reductive physicalism (psychophysical identity theory) to non-reductive physicalism (in which the mental "supervenes" on the physical) to epiphenomenalist dualism or even emergent dualism. We illustrate how these diverging interpretations result in radically different views on the identity of psychiatry and its relationship with the rapidly expanding domain of neurology/neuroscience.     

Fermented liquid feed for pigs

Since the announcement of the ban on the use of antibiotics as antimicrobial growth promoters in the feed of pigs in 2006 the investigation towards alternative feed additives has augmented considerably. Although fermented liquid feed is not an additive, but a feeding strategy, the experimental work examining its possible advantages also saw a rise. The use of fermented liquid feed (FLF) has two main advantages, namely that the simultaneous provision of feed and water may result in an alleviation of the transition from the sow milk to solid feed and may also reduce the time spent to find both sources of nutrients, and secondly, that offering FLF with a low pH may strengthen the potential of the stomach as a first line of defence against possible pathogenic infections. Because of these two advantages, FLF is often stated as an ideal feed for weaned piglets. The results obtained so far are rather variable, but in general they show a better body weight gain and worse feed/gain ratio for the piglets. However, for growing-finishing pigs on average a better feed/gain ratio is found compared to pigs fed dry feed. This better performance is mostly associated with less harmful microbiota and better gut morphology. This review provides an overview of the current knowledge of FLF for pigs,dealing with the FLF itself as well as its effect on the gastrointestinal tract and animal performance.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M “arrest,” showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53^−/− mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^−/− mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.Key words: mouse, regeneration, p53, p21, MRL, ear-hole, Tgfβ     

In vitro fermentability and physicochemical properties of fibre substrates and their effect on bacteriological and morphological characteristics of the gastrointestinal tract of newly weaned piglets

Fermentability of fibre has a great impact on the bacterial flora along the gastrointestinal tract of newly weaned piglets. Therefore, this parameter was determined by incubating in vitro different fibre substrates (chicory roots, sugar beet pulp, wheat bran and corn cobs) with contents of jejunum or caecum sampled from slaughtered pigs. Incubating with small intestinal contents, lactic acid was the only fermentation product. Fermentability was highest for chicory roots, followed by wheat bran and sugar beet pulp, while corn cobs were not fermented. Based on SCFA formed in the incubations with caecal contents, ranking of the fermentability of the fibre substrates was in the same order. The effect of adding different fibre substrates to diets of newly weaned piglets on bacteriological and morphological aspects of the gastrointestinal tract was also investigated. In Experiment 1 three groups of five piglets, weaned at four weeks of age, received a control feed (C), C supplemented with corn cobs (50 g/kg) or with chicory roots (20 g/kg). In Experiment 2, diet C was supplemented with sugar beet pulp (120 g/kg) or with wheat bran (75 g/kg). After three weeks animals were euthanized and digesta were sampled from stomach, proximal and distal jejunum, caecum and colon. Furthermore, mucosal scrapings were prepared and tissue samples were taken from jejunum, caecum and colon. Viscosity was determined for jejunal, caecal and colon contents. Corn cobs in the feed increased the number of total bacteria, lactobacilli and bifidobacteria in the stomach and proximal duodenum, while a decreased count of streptococci in distal jejunum contents was noted. Chicory roots increased the counts of Escherichia coli in the distal jejunum and on the mucosa, while sugar beet pulp decreased the number of lactobacilli on the mucosa only. Wheat bran seemed to increase the count of E. coli in jejunal digesta and on the mucosa, and also the number of lactobacilli in the stomach and jejunum. Bifidobacterial numbers were increased but only in the proximal part of the jejunum. Fibre substrates affected the concentration of lactate and SCFA in different parts of the intestinal tract. Feeding corn cobs increased villus length in the proximal jejunum by 13%. The number of intra-epithelial lymphocytes in the villous epithelium of proximal and distal jejunum was decreased by corn cobs and chicory roots supplementation while beet pulp and wheat bran had the opposite effect. In Experiment 1, apoptotic index of the mucosa of the distal jejunum was very low and decreased when corn cobs were fed. Mitotic index in the crypts was only affected by the wheat bran diet and a small decrease was noted. It was concluded that the fermentability of fibre was not an ideal criterion for predicting its effects on the flora. The effect of fibres on viscosity of digesta was negligible probably explaining the lack of clear and consistent influences on the intestinal mucosa.     

Analysis of the pattern of gene expression during human adipogenesis by DNA microarray

High density DNA microarrays containing over 5000 cDNA clones were used to carry out a comprehensive investigation of gene expression during adipogenesis. Complex probes synthesized from total RNA were hybridized to the arrays to determine the level of mRNA expression of each arrayed gene. Thirty three genes (29 known and 4 ESTs with no identified homologies) have been found to alter their level of expression more than 2.5-fold after differentiation. The quantitative measurement by DNA array was in good agreement with conventional Northern blot analysis of selected genes. Our results demonstrate that utilization of a DNA array is a speedy, efficient and quantitative approach to profile the expression of a large number of genes.