Pulmonary arterial hypertension: a disease of tethers, SNAREs and SNAPs?

Histological and electron microscopic studies over the past four decades have highlighted "plump," "enlarged" endothelial, smooth muscle, and fibroblastic cellular elements with increased endoplasmic reticulum, Golgi stacks, and vacuolation in pulmonary arterial lesions in human and in experimental (hypoxia and monocrotaline) pulmonary arterial hypertension. However, the contribution of disrupted intracellular membrane trafficking in the pathobiology of this disease has received insufficient attention. Recent studies suggest a pathogenetic role of the disruption of intracellular trafficking of vasorelevant proteins and cell-surface receptors in the development of this disease. The purpose of this essay is to highlight the molecular regulation of vesicular trafficking by membrane tethers, SNAREs and SNAPs, and to suggest how their dysfunction, directly and/or indirectly, might contribute to development of pulmonary arterial hypertension in experimental models and in humans, including that due to mutations in bone morphogenetic receptor type 2.     

Detection of proteins and bacteria using an array of feedback capacitance sensors

An integrated array of micron-dimension capacitors, originally developed for biometric applications (fingerprint identification), was engineered for detection of biological agents such as proteins and bacteria. This device consists of an array of 93,184 (256 x 364) individual capacitor-based sensing elements located underneath a thin (0.8 microm) layer of glass. This glass layer can be functionalized with organosilane-based monolayers to provide groups amenable for the immobilization of bioreceptors such as antibodies, enzymes, peptides, aptamers, and nucleotides. Upon functionalization with antibodies and in conjunction with signal amplification schemes that result in perturbation of the dielectric constant around the captured antigens, this system can be used as a detector of biological agents. Two signal amplification schemes were tested in this work: one consisted of 4 microm diameter latex immunobeads and a second one was based on colloidal gold catalyzed reduction of silver. These signal amplification approaches were demonstrated and show that this system is capable of specific detection of bacteria (Escherichia coli) and proteins (ovalbumin). The present work shows proof-of-principle demonstration that a simple fingerprint detector based on feedback capacitance measurements can be implemented as a biosensor. The approach presented could be easily expanded to simultaneously test for a large number of analytes and multiple samples given that this device has a large number of detectors. The device and required instrumentation is highly portable and does not require expensive and bulky instrumentation because it relies purely on electronic detection.     

Taxonomic importance of seed macro‐ and micro‐morphology in Abelmoschus (Malvaceae)

Seed morphology of Abelmoschus is known to be variable, but patterns of variation have never been critically studied. We studied seed macro‐ and micro‐morphological characters, including seed shape/size, seed coat pattern and trichome density/structure in multiple samples to evaluate the taxonomic significance of seed characters. Among the studied characters, seed shape and trichome structure were found to have major taxonomic importance and proved to be valuable characters for separating taxa. Two main seed types were present: seeds with deciduous trichomes and seeds with persistent trichomes. These characters offer significant evidence to the distinctness of certain species (A. esculentus, A. moschatus subsp. moschatus, A. moschatus subsp. tuberosus, A. crinitus and A. angulosus). Further, our results indicate that A. moschatus subsp. tuberosus should be maintained as a separate subspecies while A. manihot subsp. tetraphyllus var. pungens may be merged in A. angulosus. No significant intraspecific variation was observed, except in A. esculentus. We conclude that seed coat sculpturing and seed trichomes do indeed provide stable and diagnostic characters for many morphologically closely related taxa of Abelmoschus and that LM/SEM techniques can be useful in solving systematic problems and management of Abelmoschus genetic resources.     

Comparison of the anti-influenza virus activity of cyclopentane derivatives with oseltamivir and zanamivir in vivo

Cyclopentane derivatives, designated as BCX-1812, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with oseltamivir carboxylate and zanamivir for the in vivo activity in mice infected with A/Turkey/Mas/76 X A/Beijing/32/92 (H6N2) influenza virus. The compounds were tested orally and intranasally at different dose levels. BCX-1812, BCX-1827, and BCX-1923 showed more than 50% protection at 1mg/kg/day dose level on oral treatment. The intranasal treatment was 100% effective even at 0.01 mg/kg/day for all four compounds. On comparison with oseltamivir carboxylate and zanamivir, these four cyclopentane derivatives have shown equal or better efficacies. The synthesis of two new compounds, BCX-1898 and BCX-1923, is also described.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Effect of different temperature on starch synthase activity in excised grains of wheat cultivars

Excised grains of wheat (Triticum aestivum) varieties HD 2285 (relatively tolerant) and HD 2329 (susceptible type) were incubated for 1 hr at 15 degrees, 25 degrees, 35 degrees and 45 degrees C. In an another treatment, excised grains were incubated for 1 hr at increasing temperature (15 degrees, 25 degrees, 35 degrees and 45 degrees C) continuously, thus exposing the grains to gradual rise in temperature. The above treated grains were then analysed for the activity of soluble starch synthase (SSS) and granule bound starch synthase (GBSS) assayed at 20 degrees C. SSS activity decreased as the pre-exposure temperature was higher, though the tolerant variety showed lesser decrease. Decrease in SSS activity was lesser when excised grains were exposed to gradual rise in temperature from 15 degrees to 45 degrees C as compared to direct exposure to 45 degrees C. Pre-exposure of excised grains to different temperatures however, had no significant effect on GBSS activity.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

Effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drops through excised goat, sheep, and buffalo corneas

The purpose of this investigation was to evaluate the effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drop through freshly excised goat, sheep, and buffalo corneas. Aqueous isotonic ophthalmic solutions of moxifloxacin hydrochloride of different concentrations (pH 7.2) or 0.5% (wt/vol) solutions of different pH or 0.5% solutions (pH 7.2) containing different preservatives were made. Permeation characteristics of drug were evaluated by putting 1 mL formulation on freshly excised cornea (0.50 cm²) fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor (containing 10 mL bicarbonate ringer at 37°C under stirring) by spectrophotometry at 291 nm, after 120 minutes. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but a decrease in percentage permeation. Increase in pH of the solution from 5.5 to 7.2 increased drug permeation, indicating pH-dependent transport. Compared with control formulation, moxifloxacin 0.5% (wt/vol) solution (pH 7.2) containing disodium edetate (EDTA) (0.01% wt/vol) produced significantly (P<.05) higher permeation with all the corneas. Formulation with benzyl alcohol significantly (P<.05) increased permeation with buffalo cornea compared with its control. Presence of benzalkonium chloride (BAK) (0.01% wt/vol) and EDTA (0.01% wt/vol) in the formulation increased permeation to the maximum with all the corneas. The results suggest that moxifloxacin 0.5% ophthalmic solution (pH 7.2) containing BAK (0.01%) and EDTA (0.01%) provides increased in vitro ocular availability through goat, sheep, and buffalo corneas. Published: February 10, 2006 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, Sector III, New Delhi-110017, India