How Do Different Watering Regimes Affect the Growth,Chlorophyll Fluorescence,Phytohormone, and Phenolic Acid Content of Greenhouse-Grown Ceratotheca triloba?

We evaluated the effect of different watering regimes on the growth, chlorophyll fluorescence, phytohormones, and phenolic acids in Ceratotheca triloba (Bernh.) Hook.f., a commonly consumed African indigenous leafy vegetable. The study was conducted in the greenhouse under different watering regimes [seven (daily); three (thrice); two (twice); one (once) day(s) per week] for a period of 2 and 4-months. In each pot (7.5 cm diameter; 150 ml volume), 50 ml of water was applied per treatment. At the end of the experiment, plant growth, chlorophyll fluorescence, phytohormones, and phenolic acids were determined. A decrease in water availability resulted in a consistent decline in plant growth after a 4-month growth period. The severity of reduced water availability was more noticeable in plants watered once a week with a 1.4-fold reduction in growth and quantum efficiency of PSII (Fv/Fm) value of 0.80. The significant decline in growth and chlorophyll fluorescence was probably due to the increased production of abscisic acid (ABA) and cytokinin (CK) content together with the detected phytohormones in plants with restricted water supply. Furthermore, plants watered once a week had a trade-off between growth and phenolic acid production, with significantly higher (threefolds) concentrations of vanillic, ferulic, caffeic, and 4-coumaric acids in 4-month-old plants. Even though C. triloba grew best in well-watered soil, the plant had the potential to adapt and survive in soils with limited water supply for longer periods of growth. These findings suggest that regulation of phytohormones and phenolic acids played an important role in improving the growth of C. triloba under limited water conditions.

     

Immunohistochemical evidence for the expression and induction of paraoxonase in rat liver, kidney, lung and brain tissue. Implications for its physiological role

Studies on the localization of paraoxonases (PON's) are of interest because of its involvement in both the detoxication of activated organophosphorus pesticides and in the prevention of peroxidative damage to phospholipids and cholesteryl-esters in LDL and HDL particles and cell membranes during the atherogenic process. In the present study, we have investigated the cellular localization of PON1 by immunohistochemistry in different rat tissues. The protein was mainly detected in the endothelial lining of every tissue studied (liver, kidney, lung and brain). Besides, it was found in hepatocytes from the centrolobular region of the liver, in the glomeruli and basal pole of the proximal convoluted tubule of the kidney, in cells from bronchiolar epithelium and type I pneumocytes of the lung, and in leptomeningeal cells, ependymal cells and ventricular side of choroid plexus cells of the brain. However, neurons and glia lacked immunostaining. After 3-methylcholanthrene induction an increase in the intensity of immunostaining was observed in the same areas, as well as an additional staining in midzonal hepatocytes. On the basis of the tissue distribution observed for PON1, it is proposed that this enzyme might have a function related to the inactivation of oxidative stress by-products (either at a cellular level or blood-vessel wall) and other environmental chemicals. At present it has not yet been established whether the paraoxonase detected in the various tissues is truly a product of the PON1 gene or could represent products of the PON2 or PON3 genes.     

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

Rapid quantitative detection of Listeria monocytogenes in meat products by real-time PCR

We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.     

Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).     

Increased behavioural activity of rats in forced swimming test after partial denervation of serotonergic system by parachloroamphetamine treatment

The present study aimed at characterizing the effect of partial 5-HT denervation by parachloroamphetamine (PCA), a 5-HT selective neurotoxin, on forced swimming behaviour and monoamine levels in several rat brain regions. PCA was administered intraperitoneally in two independent experiments in doses of 2, 4 and 6 mg/kg and in doses 1, 2, 4 mg/kg, respectively. PCA (2 mg/kg) reduced immobility in the forced swimming test in the Experiment 1 and according to Experiment 2 this is explained by increased swimming time. Dose-dependent reductions in 5-HT and 5-HIAA levels were found in all brain regions studied, and the maximal effects were of a similar magnitude. In septum, the effect of PCA took more time to develop. The effects of the lowest dose of PCA suggest that the neurotoxin affects not only the dorsal raphe projection areas but also the fine axons which arise from the median raphe. alpha2-Adrenoceptors and beta-adrenoceptors in cerebral cortex were not affected by the PCA treatment. Binding affinity of the 5-HT(1A) receptors was higher after all doses of PCA. On the second exposure to the forced swimming the time spent in swimming was found to be negatively and the time spent in immobile posture positively correlated with serotonin turnover in frontal cortex. The time spent in struggling on the second exposure to test was found to be negatively correlated with KD of beta-adrenoceptor binding in cerebral cortex. These data suggest that partial 5-HT denervation with low doses of PCA, which elicits a specific pattern of neurodegeneration, results in an increased behavioural activity, and that the traditional interpretation of the measures in forced swimming test, despite of the test's predictive power in revealing antidepressants acting on monoaminergic systems, is not adequate for studies on the neurochemical basis of depression.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane

Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon.     

Topology of the membrane-associated hepatitis C virus protein NS4B

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in FLAVIVIRIDAE: