Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Nuclear translocation of p65 NF-kappaB is sufficient for VCAM-1, but not ICAM-1, expression in TNF-stimulated smooth muscle cells: Differential requirement for PARP-1 expression and interaction

Although nuclear translocation of NF-kappaB and subsequent binding to promoters of ICAM-1 and VCAM-1 have been shown to be decisive for their expression, a number of discrepancies in the expression patterns of these adhesion molecules have been reported in both cell culture systems and disease settings, including atherosclerosis, asthma, and autoimmune diseases. Here we show that while p65 NF-kappaB nuclear translocation in TNF-treated smooth muscle cells (SMCs) was sufficient for the expression of VCAM-1, expression of ICAM-1 showed a critical requirement for PARP-1. I-kappaBalpha phosphorylation and subsequent degradation were virtually identical in both TNF-treated wild-type and PARP-1-/- SMCs. VCAM-1 expression in TNF-treated PARP-1-/- SMCs was completely inhibited by the NF-kappaB inhibitor, pyrrolidine dithiocarbamate, confirming that VCAM-1 expression was indeed NF-kappaB-dependent. The expression of both VCAM-1 and ICAM-1 was associated with a transient interaction between PARP-1 and p65 NF-kappaB when examined in the fibroblastic cell line, COS-7, and in the airway epithelial cell line, A549. Such interactions were confirmed using florescence resonance energy transfer analysis. Protein acetylation activity, mediated by p300/CBP, was required for both VCAM-1 and ICAM-1 expression in TNF-treated SMCs; however, the interaction of PARP-1 with p300/CBP was dispensable for VCAM-1 expression. These findings indicate that p65 NF-kappaB nuclear translocation may be sufficient for certain genes (e.g., VCAM-1) while insufficient for others (e.g., ICAM-1), thus providing a novel insight into the role of NF-kappaB in driving target gene expression. Furthermore, the data suggest a differential requirement for PARP-1 expression in inflammatory processes.     

Ginkgolides protect against amyloid-β1–42-mediated synapse damage in vitro

Background

The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ_1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ_1–42 were examined.

Results

The addition of Aβ_1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ_1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ_1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ_1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ_1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ_1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E₂.

Conclusion

Pre-treatment with ginkgolides A or B protects neurons against Aβ_1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E₂, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.     

Design,Synthesis, Cytotoxic Screening and Molecular Docking Studies of Novel Hybrid Thiosemicarbazone Derivatives as Anticancer Agents

Thiosemicarbazones have been the focus of scientists owing to their broad clinical anticancer range. Herein, A Series of new thiosemicarbazone derivatives 5 – 9 were synthesized and confirmed through the use of different spectroscopic techniques along with elemental analysis. The in vitro cytotoxic activity of compounds 5 – 9 against MCF-7 and A549 cell lines and normal breast cells were assessed. Several compounds were found to be active. The most active compound 7 caused MCF-7 cell cycle arrest at G1/ S phases; and induced apoptosis at the pre-G1 phase. The apoptosis-inducing activity of compound 7 was proofed by the elevation of caspase 3/7 activity and also by up-regulation of the expression of Bax and p53 proteins together with the down-regulation of the expression of the Bcl-2 protein. It also had a strong inhibitory effect topoisomerase IIβ enzyme. Molecular Docking study revealed that the synthesized compounds had good docking scores compared to the standard drug Etoposide towards the topoisomerase IIβ protein (3QX3). Overall, these findings confirmed that the new thiosemicarbazone derivatives could aid in the development of promising cancer drug candidates.     

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.     

Assessing the environmental profile of orange production in Brazil

Background, aim, and scope

Brazil is the world’s largest orange producing country, with a total planted area of more than 820,000 hectares. The bulk of the oranges produced in Brazil (70%) is processed into frozen concentrated orange juice (FCOJ) by large processing companies. Exports represent around 97% of the total FCOJ produced, making Brazil the largest world producer and exporter of FCOJ. The Brazilian citrus sector accounts for half of the world’s supply of orange juice and for 80% of the juice traded on the international market. The goal of this study is to characterize the Brazilian orange producers in terms of farm size, cultivated varieties, watering system and tillage practices. In addition, the study presents some aspects of the LCI of oranges grown specifically for the production of FCOJ in the two major orange-growing areas in Brazil (the Northern and Southern regions of the State of São Paulo) during reference crop year 2002/03 in order to generate detailed production inventory data and identify the potential environmental impacts of tillage, both of which are key to enable the formulation of sustainability parameters for the production of FCOJ in Brazil.

Methods

This study was performed in compliance with the guidelines and requirements of the ISO 14040 standard series. All information and data considered and evaluated in this study (the use of water, energy, fertilizers, pesticides and soil correctors) were gathered through in-depth questionnaires either filled in directly by the farm manager or completed by the farm manager and sent in by mail. The data cover a total of 367,200 metric tons of oranges produced by 4 million plants in commercial production from a total evaluated area that accounts for 19.5% of the overall production of oranges in the State of São Paulo. The two major orange-producing areas in Brazil located respectively in the Northern and Southern regions of the State of São Paulo were evaluated. Only the inputs and outputs associated with tillage practices and technology were considered in this paper. The environmental aspects of the production of fertilizers and pesticides were not included within the boundaries. The functional unit selected for this study was 1,000 kg of oranges for FCOJ. Farm-specific data have been combined with agricultural production data to construct an orange cultivation model.

Results

The orange varieties for the production of FCOJ evaluated in this study were Pêra, Valência and Natal. The farms investigated had a cultivated area varying from 22 to 7,000 ha, with a plant density ranging from 170 to 500 plants/ha and an average yield of 30,500 kg/ha for mature trees. Depending on the region, the production of 1,000 kg of oranges requires approx. 120 to 4,400 MJ of energy; 0.3 to 36 kg of diesel; 1,100 to 54,500 kg of water for irrigation; 0.3 to 65 kg of fertilizers (NPK); 0.1 to 13.5 kg of pesticides; 8 to 650 kg of soil correctors. The distribution of some inputs across the farms located in the two main Brazilian orange-producing regions is presented, in addition to the total weighted average.

Discussion

In addition to the variations among the regions evaluated, it was generally observed that there were very considerable differences in order of magnitude between the data collected from farms located in the same region. Although the inputs are directly related to the specific characteristics of each farm and the climatic and topographic conditions of its location, this study has identified some farms that are in a good position to reduce the amounts of some inputs and enhance their environmental and economical performance.

Conclusions

Taking into account the aspects evaluated in this study, only 21% of the orange growers showed good performance, i.e., consumption of pesticides, fertilizers and soil correction compounds equal to or lower than the weighted averages, except for “land use”, which was above the average for the majority of these farms. This study provided important results that allow for a better understanding of the agricultural practices involved and the potential environmental impacts of this product.

Recommendation and perspective

The authors would like to suggest that the five best-performing farms average values be set as the maximum allowable level and point of departure for the discussion on the good environmental performance of Brazilian FCOJ. Besides the total inputs, the role of good agricultural practices to achieve good yields and, at the same time, improve the sustainability of orange growing should also be taken into consideration. Future updates of this study will show the evolution of natural resource management as a result of improved land use, new agricultural practices, reduced use of fertilizers and agrochemicals.     

Functional interaction of CD154 protein with α5β1 integrin is totally independent from its binding to αIIbβ3 integrin and CD40 molecules

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ～4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Radial basis function neural networks for modeling growth rates of the basidiomycetes Physisporinus vitreus and Neolentinus lepideus

A radial basis function (RBF) neural network was developed and compared against a quadratic response surface (RS) model for predicting the specific growth rates of the biotechnologically important basidiomycetous fungi, Physisporinus vitreus and Neolentinus lepideus, under three environmental conditions: temperature (10–30 °C), water activity (0.950–9.998), and pH (4–6). Both the RBF network and polynomial RS model were mathematically evaluated against experimental data using graphical plots and several statistical indices. The evaluation showed that both models gave reasonably good predictions, but the performance of the RBF neural network was superior to that of the classical statistical method for all three data sets used (training, testing, full). Sensitivity analysis revealed that of the three experimental factors the most influential on the growth rate of P. vitreus was water activity, followed by temperature and pH to a lesser extent. In contrast, temperature in particular and then water activity were the key determinants of the development of N. lepideus. RBF neural networks could be a powerful technique for modeling fungal growth behavior under certain parameters and an alternative to time-consuming, traditional microbiological techniques.     

Detection of a fourth orbivirus non-structural protein

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1-VP7) and 3 non-structural proteins (NS1-NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.