Substance P-Evoked Calcium Mobilization and Ionic Current Activation in the Human Astrocytoma Cell Line U 373 MG: Pharmacological Characterization

Abstract— In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage-clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [l -Pro⁹]SP displayed high potencies in both assays with EC₅₀values of 2.5 ± 10^?9M and 1 ± 10^?9M on calcium responses and 110^?9M and 510^?9M on ion current responses, respectively. The high potency of SP and [l -Pro⁹]SP as well as the low potency of [Lys⁵,MeLeu⁹,N-Leu¹⁰]neurokinin A_(4-10) and the inactivity of senktide demonstrate the NK₁-type pharmacology of these responses. Furthermore, the NK₁ antagonists (±)-CP 96,345, its chloro analogue, (±)-cis-3-(2-chlorobenzylamino)-2-benz-hydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (1 (10^?7M), the IC₅₀ values for the three antagonists were 4 ± 10^?10M, 4 ± 10^?9M, and 9 ± 10^?9M, respectively, whereas on the current response evoked by SP 10^?8M), the IC₅₀ values were 8 ± 10^?9M, 2.4 ± 10^?8M, and 1.2 10^?7M, respectively. Despite differences in the absolute IC₅₀ values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well. The U 373 MG cell line provides a useful model system for studies of the pharmacology of the human NK₁receptor and its transduction mechanisms at the level of second messengers and modulation of ion currents.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions

Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

5-Lipoxygenase as a drug target: A review on trends in inhibitors structural design,SAR and mechanism based approach

The most common inflammatory disease of the airways is asthma among children affecting around 235 million people worldwide. 5-Lipoxygenase (5-LOX) is a crucial enzyme which helps in the conversion of arachidonic acid (AA) to leukotrienes (LTs), the lipid mediators. It is associated with several inflammation related disorders such as asthma, allergy, and atherosclerosis. Therefore, it is considered as a promising target against inflammation and asthma. Currently, the only drug against 5-LOX which is available is Zileuton, while a few inhibitors are in clinical trial stages such as Atreleuton and Setileuton. So, there is a dire requirement in the area of progress of novel 5-LOX inhibitors which necessitates an understanding of their structure activity relationship and mode of action. In this review, novel 5-LOX inhibitors reported so far, their structural design, SAR and developmental strategies along with clinical updates are discussed over the last two decades.