Induction of T-cell proliferation and enhancement of NK activity by supernatants from Con A-stimulated human peripheral blood mononuclear cells: a new lymphokine

Supernatants from human peripheral blood mononuclear cells activated by Con A contain a factor(s) that stimulates blastogenic activity of normal human peripheral blood mononuclear cells. This Con A supernatant (CAS) contains stimulatory activity for E-rosette positive lymphocytes (T cells) and requires adherent cells for stimulation of T-cell proliferation. CAS does not contain detectable amounts of IL-2 as determined by its inability to support CTLL cell growth. Nor does it contain IL-1 or interferon. Examination of functional activity of lymphocytes stimulated for 3 days by CAS revealed that NK activity is augmented. This supernate does not appear to have any direct effect on B-cell function, although it induces suppression of polyclonal PWM stimulation of immunoglobulins. Thus, CAS appears to contain a new cytokine with immunomodulating potential.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

Identification of a mutation associated with permethrin resistance in the para-type sodium channel of the stable fly (Diptera: Muscidae)

The insect sodium channel is of particular interest for evaluating resistance to pyrethroids because it is the target molecule for this major class of neurotoxic insecticides. The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), sodium channel coding sequence representing domains IS6 through IVS6 was isolated, and the sequence encoding domain II was compared among individuals of a laboratory strain selected for resistance to permethrin and the unselected, parental generation. A point mutation resulting in a leucine-to-histidine amino acid change was identified (Leul014His), and its location corresponded with that observed for knockdown resistance (kdr) mutations in other insects. As a result, the allele was designated kdr-his. A molecular assay was developed to assess the frequency of this mutation in genomic DNA of individual stable flies from the laboratory selections, which provided further evidence that the kdr-his allele accounts for the observed level ofpermethrin resistance in the selected strain. The assay was then used to evaluate the frequency of the mutation from five field-collected populations originating from three horse farms near Ocala, FL; one horse farm near Gainesville, FL; and one dairy farm near Hague, FL. Frequency of the kdr-his allele ranged from 0.46 to 0.78, supporting further investigation of allele prevalence throughout the stable fly season and in response to field insecticide application.     

Positive Reinforcement Training in Squirrel Monkeys Using Clicker Training

Nonhuman primates in research environments experience regular stressors that have the potential to alter physiology and brain function, which in turn can confound some types of research studies. Operant conditioning techniques such as positive reinforcement training (PRT), which teaches animals to voluntarily perform desired behaviors, can be applied to improve behavior and reactivity. PRT has been used to train rhesus macaques, marmosets, and several other nonhuman primate species. To our knowledge, the method has yet to be used to train squirrel monkeys to perform complex tasks. Accordingly, we sought to establish whether PRT, utilizing a hand‐box clicker (which emits a click sound that acts as the conditioned reinforcer), could be used to train adult male squirrel monkeys (Saimiri boliviensis, N = 14). We developed and implemented a training regimen to elicit voluntary participation in routine husbandry, animal transport, and injection procedures. Our secondary goal was to quantify the training time needed to achieve positive results. Squirrel monkeys readily learned the connection between the conditioned reinforcer (the clicker) and the positive reinforcer (food). They rapidly developed proficiency on four tasks of increasing difficulty: target touching, hand sitting, restraint training, and injection training. All subjects mastered target touching behavior within 2 weeks. Ten of 14 subjects (71%) mastered all tasks in 59.2 ± 2.6 days (range: 50–70 days). In trained subjects, it now takes about 1.25 min per monkey to weigh and administer an intramuscular injection, one‐third of the time it took before training. From these data, we conclude that clicker box PRT can be successfully learned by a majority of squirrel monkeys within 2 months and that trained subjects can be managed more efficiently. These findings warrant future studies to determine whether PRT may be useful in reducing stress‐induced experimental confounds in studies involving squirrel monkeys. Am. J. Primatol. 74:712–720, 2012. © 2012 Wiley Periodicals, Inc.     

Enhanced cellulase production by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> by cultivation on glycerol followed by induction on cellulosic substrates

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.     

Competency for graviresponse in the leaf-sheath pulvinus of Avena sativa: onset to loss

The development of the leaf-sheath pulvinus of oat (Avena sativa L. cv. Victory) was studied in terms of its competency to respond to gravistimulation. Stages of onset of competency, maximum competency and loss of competency were identified, using the length of the supertending internode as a developmental marker. During the early phases in the onset of competency, the latency period between stimulus and graviresponse decreased and the steady state response rate increased significantly. When fully competent, the latency period remained constant as the plant continued to develop, suggesting that the latency period is relatively insensitive to quantitative changes (e.g., in carbohydrate or nutrient availability) at the cell level within the plant. In contrast, the response rate was found to increase with plant development, indicating that graviresponse rate is more strongly influenced by quantitative cellular changes. The total possible graviresponse of a single oat pulvinus was confirmed to be significantly less than the original presentation angle. This was shown to not result from a loss of competency, since the graviresponse could be reinitiated by increasing the presentation angle. As a result of the low overall graviresponse of individual pulvini, two or more pulvini are required to bring the plant apex to the vertical. This was determined to occur though the sequential, rather than simultaneous, action of successive pulvini, since a given pulvinus lost competency to gravirespond shortly after the next pulvinus became fully competent.     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings

Etiolated Arabidopsis thaliana seedlings, lacking a functional prephenate dehydratase1 gene (PD1), also lack the ability to synthesize phenylalanine (Phe) and, as a consequence, phenylpropanoid pigments. We find that low doses of ultraviolet (UV)-C (254 nm) are lethal and low doses of UV-B cause severe damage to etiolated pd1 mutants, but not to wild-type (wt) seedlings. Furthermore, exposure to UV-C is lethal to etiolated gcr1 (encoding a putative G protein-coupled receptor in Arabidopsis) mutants and gpa1 (encoding the sole G protein alpha subunit in Arabidopsis) mutants. Addition of Phe to growth media restores wt levels of UV resistance to pd1 mutants. The data indicate that the Arabidopsis G protein-signalling pathway is critical to providing protection from UV, and does so via the activation of PD1, resulting in the synthesis of Phe. Cotyledons of etiolated pd1 mutants have proplastids (compared with etioplasts in wt), less cuticular wax and fewer long-chain fatty acids. Phe-derived pigments do not collect in the epidermal cells of pd1 mutants when seedlings are treated with UV, particularly at the cotyledon tip. Addition of Phe to the growth media restores a wt phenotype to pd1 mutants.