Different vanilloid agonists cause different patterns of calcium response in CHO cells heterologously expressing rat TRPV1

The vanilloid receptor subtype 1 (TRPV1 or VR1) is expressed in nociceptive primary afferents of the C-fiber 'pain' pathway and has attracted considerable attention as a therapeutic target. Here, using rat TRPV1 heterologously expressed in Chinese hamster ovary cells, we show that different agonists show different patterns of modulation of the intracellular Ca2+ concentration, monitored in individual cells by fura-2 Ca2+ imaging. We identified 5 parameters (potency, maximal response, latency of response, variability of latency of response among individual cells, and desensitization) which behaved differently for different compounds. The potencies of the compounds examined ranged from EC50 values of 80 pM to 9 microM. Peak levels of induced [Ca2+]i were observed either higher (RTX) or lower (anandamide) than for capsaicin. Significant latencies of response were observed for some (e.g. RTX) but not other derivatives, with great variation among individual cells in this latency. Marked desensitization after stimulation was detected in some cases (e.g. anandamide, capsaicin); for others, no desensitization was observed. We conclude that structurally diverse vanilloid agonists induce marked diversity in the patterns of Ca2+ response. This diversity of response may provide opportunities for pharmacological exploitation.     

Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus

To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped with the 6xHis tag, a simple colony-blot procedure can be performed and a colony giving strong and efficient expression can easily be selected for high range protein expression. We utilized this procedure to produce one of potato mop-top virus (PMTV) movement proteins, namely triple gene block protein 1 (TGBp1) which was very hard to express in bacteria in its original length. The TGBp1 gene was digested with exonuclease III and nuclease S1 from its 5' terminus, leaving 6xHis tag intact. The clone that showed the strongest signal with anti-His antibodies in colony-blot procedure was found to have 44 amino acids (of total 463) deleted. The SDS-PAGE and Western blot of high range bacterial culture lysate confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.     

Conformation dependence of the CαDα stretch mode in peptides: Side‐chain influence in dipeptide structures

We have shown by theoretical studies of alanine peptides that the C^αD^α stretch frequency could be particularly useful for determining peptide structure because of its sensitivity to the φ,ψ torsion angles at the C^α atom. To demonstrate that this is a robust methodology worthy of experimental exploration, we have also shown that this mode is even more determinative of conformation in aqueous solution, mainly as a result of the development of differential C^α? D^α···O(water) interactions. As further assurance, we now determine the influence of the side chain on this band, showing for aliphatic, a polar, and an aromatic side chains that the dependence is minor and explaining why this is also expected for other side chains. These results should stimulate new experimental methodologies in the field of peptide structure determination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1065–1071, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Role of the C8 gem-dimethyl group of bryostatin 1 on its unique pattern of biological activity

The role of the C(8) gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.     

Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state

CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF?) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF?-coated 96-well plates and cultured under na?ve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF?-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF?-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.     

New and little known cladocerans (Crustacea: Anomopoda) from southeastern Mexico

Mexico represents a transitional area in which two biogeographic regions meet. A survey of the cladocerans collected in true tropical waterbodies from the country revealed a diverse fauna. The environments examined included temporary pools in the forest, rivers, permanent lagoons and karstic sinkholes. New records or little known taxa include: Diaphanosoma brevireme, D. fluviatile, Pseudosida ramosa, Simocephalus mixtus, Onchobunops tuberculatus,Guernella raphaelis, Chydorus nitidilus, Notoalonacf. globulosa, Alonella brasiliensis and Leydigiopsis curvirostris. In contrast to high altitude systems, with clear nearctic affinity, the lowlands of southeastern Mexico showed a striking resemblance with the South and Central American cladoceran fauna. Apparently, orographic barriers control the distribution of the cladoceran taxa in this region.     

First report of <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> and <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> as phosphorus solubilizers in maize

Maize is one of the most important crops worldwide. It provides food for humans and animals and is used in biotechnological and industrial processes to produce a wide variety of products. The phosphorus (P) requirement for its development and production is high, but the absorption efficiency of this nutrient is insufficient to meet its requirements. The use of P-solubilizing fungi can increase this efficiency, thus reducing the amount of fertilizers applied to the crops. Therefore, this study aimed to verify the potential use of A. sydowii and A. brasiliensis and their effect in the field as bioinoculants when associated with three P fertilization doses. The experiment was carried out in a maize field, and treatments were presence and absence of fungi associated with P mineral fertilization doses. The parameters evaluated were shoot dry matter, P content in the plant, and P content in the soil. A. sydowii caused the highest P content in the plant and soil at fertilization doses of 75% and 100%, contradicting the expected results from in vitro assays that indicated that A. brasiliensis would provide better rates of P uptake. There was no difference in the other fertilization doses or for dry matter when comparing all treatments. This result strongly suggests that the use of A. sydowii can improve the efficiency of P absorption with fertilization application. In addition, the molecular analysis of the two fungi performed in this study contributes novel information on the use of both species during the agricultural process.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

An imbalance in autophagy contributes to retinal damage in a rat model of oxygen-induced retinopathy

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP.