Variability of low-molecular-weight serum subproteome in healthy humans under the conditions of normal vital activity

To study interindividual variability of the low-molecular-weight serum subproteome in healthy humans, the proteome profiles of blood sera were studied in subjects divided into three age groups: from 20 to 30 years (36 subjects), from 30 to 40 years (11 subjects), and from 40 to 50 years (11 subjects). Serum samples were fractionated by MB WCX magnetic beads using a ClintProt robot prior to the mass spectrometry based profiling. The mass spectra have been obtained using an Autoflex III time-of-flight mass spectrometer (Bruker Daltonics) in the automated mode. The low-molecular-weight serum subproteome in healthy humans was found to be characterized by significant interindividual variability: 21% of all the peaks in the proteome profiles had a coefficient of variation of more than 50%, and 29% of all the peaks had a low variance (CV < 30%). Therefore, the majority of the peaks in the proteome profile had a moderate group variation (the CV was in the interval from 30 to 50%). Fragments of high-molecular-weight kininogen, inter-α-trypsin inhibitor, C3 and C4a complement components, CI apolipoprotein, platelet factor IV, β2-microglobulin, and C cystatin were shown to display a wide variation among the tested groups of healthy humans. The peak area variance of high-molecular-weight kininogen, inter-α-trypsin inhibitor, AII and CIII apolipoproteins increased with age.     

Phytotoxic effects of β-pinene on early growth and associated biochemical changes in rice

β-Pinene, an oxygenated monoterpene, is one of the major monoterpenes emitted into the atmosphere from forest areas and trees. Besides, it is a principal component of essential oils of a number of aromatic plants, which are involved in a variety of ecological interactions, including allelopathy, in the natural environment. However, studies pertaining to phytotoxicity and biochemical effect(s) of β-pinene are largely lacking. We investigated the effect of β-pinene (0.02, 0.04, 0.08, 0.20, 0.40 and 0.80 mg/ml) in a dose- and time-dependent manner on early seedling growth, dry weight accumulation, photosynthetic pigments and changes in macromolecule (protein and carbohydrate) content and activities of enzymes—proteases, α- and β-amylases, polyphenol oxidases and peroxidases- in rice (Oryza sativa) after 3rd, 5th and 7th day of exposure. β-pinene (≥0.04 mg/ml) significantly reduced the root (by 13–87%) and coleoptile (by 5–80%) length of rice. Exposure to β-pinene reduced total chlorophyll content in rice coleoptiles suggesting a negative impact on photosynthesis. The content of macromolecules (proteins and carbohydrates) enhanced significantly in response to β-pinene, whereas the activities of hydrolyzing enzymes—proteases, α-amylases, and β-amylases—declined (by 30–85, 26–84, 27–74%, respectively) in β-pinene-exposed seedlings. In contrast, the activities of peroxidases (POX) and polyphenol oxidases (PPO) enhanced significantly (by 16–152 and 53–290%, respectively) in rice roots in response to β-pinene in a dose- and time-dependent manner. Increased activities of POX and PPO indicate their involvement in providing protection and/or conferring resistance against β-pinene-induced stress. The study concludes that β-pinene inhibits the early growth of rice by altering the plant biochemical status and enhancing activities of POXs and PPOs involved in general plant defense.     

Lipid synthesis in macrophages during inflammation in vivo: Effect of agonists of peroxisome proliferator activated receptors α and γ and of retinoid X receptors

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [¹⁴C]oleate incorporation into cholesteryl esters and triglycerides and [¹⁴C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of ¹²⁵I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of ¹²⁵I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 364–374.     

A mutant α-amylase with only part of the catalytic domain and its structural implication

A truncated mutant α-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type α-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (β/α)₈-barrel catalytic domain and did not have the three conserved catalytic residues of wild type α-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K _m for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30°C. In contrast, the K _m for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0–6.2 and 45–50°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of α-1,4-glucosidic bonds. T. Ke and X. D. Ma contributed equally to this work     

Antimicrobial activity of the extracts from fruits of <Emphasis Type="Italic">Rumex</Emphasis> L. species

This study was designed primarily to investigate the antibacterial and antifungal activity of the extracts from fruits of six Rumex L. species: R. acetosa L., R. acetosella L., R. confertus Willd., R. crispus L., R. hydrolapathum Huds. and R. obtusifolius L. The 7 Grampositive and 7 Gram-negative bacteria strains and 5 fungal ones were tested by agar and broth dilution method. Determination of minimal inhibitory concentration (MIC) revealed that the extracts from R. confertus, R. crispus, R. hydrolapathum and R. obtusifolius exerted differential inhibitory effect on the growth of Gram-positive bacteria — staphylococci (MIC=62.5–125 μg/mL) and Gramnegative bacteria — Escherichia coli ATCC 3521, Proteus mirabilis, Pseudomonas aeruginosa (MIC=125→500 μg/mL); MIC values determined by agar dilution method were somewhat higher. The same extracts inhibited also the growth of fungi — Candida spp. or Trichophyton mentagrophytes ATCC 9533 (MIC=250–500 μg/mL), as found by agar dilution method. The total content of polyphenols (11.66–78.36 mg/g), anthracene derivatives (0.26–12.93 mg/g) and tannins (4.00–11.16%) was also determined.     

Gene frequency-based estimation of natural outcrossing in opium poppy (<Emphasis Type="Italic">Papaver somniferum</Emphasis> L.)

Mating systems are generally thought to be important factors in determining the amount and nature of genetic variability in a population. Nearly 1,000 individuals at a single location (Lucknow) and over two years were crossed and subsequently scored for selfing versus outcrossing in 9–10 monohybrid populations of opium poppy (Papaver somniferum). Three different alleles of two marker loci (two—R/r and P/p—for anthocyanin locus and B/b for capsule size locus) were used to determine the male gametes that had effected fertilizations in F₂ recessives (rr, pp and bb). The estimates of the gene frequency-based outcrossing parameter (α) were found to vary with year, cross and marker locus used (α range: 7.21–71.03%). Study of the two dihybrid crosses concerning the two marker loci simultaneously, further confirmed that outcrossing at the R/r or P/p locus was significantly greater than that at the B/b locus. The nature of the outcrossing was, in general, nonrandom. In this species, in general, selfing predominated, with one exception in respect of monohybrid crosses involving the purple form of anthocyanin locus, in which outcrossing predominated.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Relationship between the Interhelical Packing Angles and the Length of α-Helices in Proteins

Abstract—Mutual arrangement, or packing, of α-helices in proteins depends on several factors, but, tight packing and the chemical nature of the polypeptide chain are the most important. This study shows, for the first time, that the torsion packing angles between axes of α-helices depend on their length. A database of helical pairs formed by two connected and juxtaposed α-helices has been compiled using the Protein Data Bank. These helical pairs have been subdivided into four types: (1) 10474 pairs formed by long helices; (2) 3665 pairs in which the first α-helix is long and the second is short; (3) 3648 pairs in which the first α‑helix is short and the second is long; 4) 1895 pairs in which both helices are short. Analysis of the database showed that most helical pairs in which both the helices are long form α-hairpins having interhelical packing angles of Ω ≈ 20°. Most helical pairs in which one α-helix is long and the other is short or both helices are short form αα-corners having orthogonal (Ω ≈ –70°…–90°) or slanted (Ω ≈ –50°) packing of α-helices. The possible reasons for this relationship between interhelical angles (Ω) and the length of α-helices are discussed. These results are of great importance in protein modeling and prediction since they enable the determination of the mutual arrangement of α-helices in protein molecules.     

Altered proteome profiles in maternal plasma in pregnancies with fetal growth restriction

Fetal growth restriction (FGR) affects 3–5% of pregnancies and is associated with increased perinatal morbidity and mortality. Currently, there is no reliable biochemical test to differentiate a pathological FGR from a nonpathological one. The objective of this study was to screen whole maternal plasma to identify differentially expressed relatively abundant proteins associated with FGR. We analyzed maternal plasma from FGR (n=28) and healthy (n=22) pregnancies using two-dimensional gel electrophoresis (2D-GE) followed by software image analysis. Three spots with molecular weight (M_r) 18 kDa corresponding to haptoglobin (hp) α2, as identified by LC-MS/MS and immunoblotting, showed differential expression patterns in FGR. The distribution of hp α2 variants in maternal plasma samples showed the hp α2 variant 1 was low in 72% of FGR, medium in 16%, whereas high in 12%. In comparison, hp α2 variant 1 was high in (41%) of controls, medium in 41%, and low in 18% of cases. Based on the software image analysis, the mean spot volume for hp α2 variant 1 was 0.12 (SD=0.18) for FGR compared to 0.26 (SD=0.19) for control (p=0.006). Given that hp turnover is indicative of its maturation process and is traceable in plasma by its dominant/suppressed variants, we propose that hp α2 is an important potential target for evaluation of its clinical and pathophysiological role and as a diagnostic biomarker in FGR.     

New basal media for half-anther culture of <Emphasis Type="Italic">Anthurium andreanum</Emphasis> Linden ex André cv. Tropical

A successful protocol for high frequency callus induction and plant regeneration from Anthurium andreanum Linden ex André cv. Tropical half-anthers is described. Different variables using Winarto and Teixeira and Murashige and Skoog basal media supplemented with several plant growth regulators [2,4-dichlorophenoxy acetic acid (0.1–1.0 mg/l), α-naphthalene acetic acid (0.01–0.2 mg/l), thidiazuron (0.5–2.0 mg/l), 6-benzylaminopurine (0.5–1.0 mg/l), and kinetin (0.5–1.0 mg/l)] were tested for their ability to induce high frequency callusing in half-anthers, indirect regeneration and rooting of shoots. Basal medium, as well as the combination and concentration of hormones applied, had a significant effect on callus formation, shoot regeneration and adventitious root formation. Winarto and Teixeira-1, an original basal medium containing 0.01 mg/l α-naphthalene acetic acid, 0.5 mg/l thidiazuron and 1.0 mg/l 6-benzylaminopurine was suitable for callus formation while an improved basal medium i.e., New Winarto–Teixeira-3 supplemented with 0.25 mg/l 2,4-dichlorophenoxy acetic acid, 0.02 mg/l α-naphthalene acetic acid, 1.5 mg/l thidiazuron and 0.75 mg/l 6-benzylaminopurine enhanced callus formation. High shoot regeneration and multiplication was also possible on New Winarto–Teixeira-3. Shoots formed a strong adventitious root system on New Winarto–Teixeira-3 containing 0.2 mg/l α-naphthalene acetic acid and 1.0 mg/l kinetin. Plantlets that varied in size and performance were successfully acclimatized and adapted to ex vitro conditions. Cytological analysis of 180 acclimatized-plantlets ex vitro revealed that 34 were haploid (n = 14–18), 15 aneuploid (n = 20–26), 126 diploid (n = 28–34) and 5 triploid (n = 45–57). The potential use of this protocol for developing half-anther culture of other Anthurium species or cultivars is discussed.     

Test of the electro-ultrafiltration method's applicability in soil analysis. Reproducibility of the method

Summary The applicability of the Electro-Ultra-Filtration (EUF) method in soil analyses was studied. The reproducibilities of the amounts of soil extracts, of ion concentrations in the extracts and of the distribution of cations and anions over the cathode and anode extracts by use of fully automatic EUF equipment were tested. The degree of variability among replicates was expressed as coefficient of variation (CV) and as the highest percentual divergence of an individual analytical measurement from the mean (L). The extraction volumes of five replicates of six different soils were found to vary between 1.1–7.1% with an average of 3.8%, as CV and between 1.5–11.3% as L. The reproducibility of desorbed P in the anode extract varied between 2.7–31.7% with an average of 8.7%, as CV and between 3.2–37.9% as L. Corresponding values for CV and L of K desorbed varied between 1.3–13.9% and 1.6–23.8%, respectively. Variations among replicates of desorbed P were especially high in the first 1–2 sub-fractions of a total of seven fractions in a single extraction run. Low K concentrations in the extract had a slightly negative influence on the reproducibility of K desorption. Furthermore, it was found that a portion of the cations is collected in the anode extract and a portion of the anions in the cathode extract, especially at the beginning of an extraction run. Pooling of anode and cathode extracts before analysis is therefore recommended.     

The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα was trypsinised, and the five fragments produced (spanning residues 1–14, 21–30, 31–87, 89–102 and 103–109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTα(89–102) and proTα(103–109) significantly fortified healthy donor-lymphocytes’ immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89–102) and proTα(103–109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1–14), proTα(21–30) and proTα(31–87) marginally upregulated lymphocyte activation. This is the first report showing that proTα’s immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.     

Influence of intramolecular interactions on conformational and dynamic properties of analogs of heptapeptide AFP<Subscript>14–20</Subscript>

Conformational and dynamic properties of proteins and peptides play an important role in their functioning. However, mechanisms that underlie this influence have not been fully elucidated. In the present work we computationally constructed analogs of heptapeptide AFP_14–20 (LDSYQCT) — one of the biologically active sites of human α-fetoprotein (AFP) — to study their conformational and dynamic properties using molecular dynamics simulation. Analogs were obtained by point substitutions of amino acid residues taking into account differences in their physicochemical properties and also on the basis of analysis of amino acid substitutions in the AFP_14–20-like motifs revealed in different physiologically active proteins. It is shown that changes in conformational mobility of amino acid residues of analogs are due to disruption or arising of intramolecular interactions that, in turn, determine existence of steric restrictions during rotation around covalent bonds of the peptide backbone. Substitution of an amino acid by another one with significant difference in physicochemical properties may not lead to remarkable changes in conformational and dynamic properties of the peptide if intramolecular interactions remain unchanged.     

Production of raw-starch-hydrolysing α-amylase from the newly isolated Geobacillus thermodenitrificans HRO10

An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 2⁴ and 3² factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T² − 8.448pH² + 6.020TpH − 0.005T²pH²) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important observation.     

Optimizing the biodegradation of two keratinous wastes through a Bacillus subtilis recombinant strain using a response surface methodology

Statistical optimization of the biodegradation of two keratinous wastes directed by Bacillus subtilis recombinant cells was carried out by means of a response surface methodology. A Box–Behnken design was employed to predict the optimal levels of three variables namely, keratin percent, incubation time and inoculum size. Analysis of variance revealed that, only keratin percent had the highest significant effect. Canonical analysis and ridge max analysis were used to get the optimal levels of the three predictors along with the optimum levels of the responses. The optimal sets of predicted and validated levels of the three variables were [7.69% (w/v) feathers, 96.58 h and 1.28% (v/v) inoculum size] and [8% (w/v) feathers, 98.45 h, 3.9% (v/v) inoculum size] to achieve the highest levels of soluble proteins (1.25–1.7 mg/ml) and NH₂-free amino groups (245.82–270.0 μmol leucine/ml), respectively upon using three optimized feathers-based media. These values represented 83.67–100% and 100% adequacy for the models of soluble proteins and NH₂-free amino groups, respectively. While, [8.23% (w/v) sheep wool, 5.52% (v/v) inoculum size and 46.58 h] and [8.33% (w/v) sheep wool, 5.89% (v/v) inoculum size and 63.46 h] were the optimal sets of predicted and validated levels of the above variables to achieve the highest yields of soluble proteins (3.4–4.6 mg/ml) and NH₂-free amino groups (290.9–302.0 μmol leucine/ml), respectively upon using three optimized sheep wool-based media. These values represented 100% adequacy for the models of soluble proteins and NH₂-free amino groups. By the end of the optimization strategy, a fold enhancement (2.14–2.43 and 1.78–2.12) in the levels of released soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized feathers-based media. However, a fold enhancement (4.25–5.75 and 2.42–2.5) in the levels of soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized sheep wool-based media. Data would encourage pilot scale optimization of the biodegradation of these wastes.     

Neuronal pathways in the guinea-pig lumbar sympathetic ganglia as revealed by immunohistochemistry

Summary Tyrosine hydroxylase (TH)- and peptide-immunoreactivity of postganglionic neurons and of nerve fibres in guinea pig lumbar paravertebral sympathetic ganglia 2–4 after transection of the communicating rami and the visceral branches, respectively, were investigated by single-and double-labelling techniques. Six subpopulations of postganglionic neurons were discriminated immunohistochemically: two cell types, which were immunoreactive to only one of the applied antisera — TH, and vasoactive intestinal polypeptide (VIP); and four cell types in which immunoreactivity was colocalized — TH/neuropeptide Y (NPY), NPY/VIP, dynorphin/α-neoendorphin and dynorphin (α-neoendorphin)/NPY. Small intensely fluorescent (SIF) cells dependent on their location exhibited differential immunobehaviour to NPY-/dynorphin-(α-neoendorphin-) and TH-antisera. Immunoreactivity to substance P (SP), calcitonin gene-related peptide (CGRP), met-enkephalin-arg-phe (MEAP) and leu-enkephalin was present in nerve fibres but not in postganglionic neurons with frequent colocalization of SP/CGRP- and MEAP/leu-enkephalin- and, sometimes leu-enkephalin/SP- and dynorphin/SP-immunoreactivity. TH-immunoreactive intraganglionic nerve fibres were numerically more increased after cutting the visceral branches, than after transection of the communicating rami. Vice versa, NPY-, VIP-, dynorphin- and α-neoendorphin-immunoreactive nerve fibres were particularly increased in number after cutting the communicating rami. Many but not all of the nerve fibres exhibited colocalization of two of these peptides. SP-, CGRP-, and enkephalin-immunoreactive nerve fibres were not visibly affected by cutting the visceral branches but virtually disappeared after lesioning the communicating rami.     

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l⁻¹) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l⁻¹) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l⁻¹ TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.     

Development and Evaluation of Buccal Bioadhesive Tablet of an Anti-emetic Agent Ondansetron

The aim of the present study was to develop and evaluate a buccal adhesive tablet containing ondansetron hydrochloride (OH). Special punches and dies were fabricated and used while preparing buccal adhesive tablets. The tablets were prepared using carbopol (CP 934), sodium alginate, sodium carboxymethylcellulose low viscosity (SCMC LV), and hydroxypropylmethylcellulose (HPMC 15cps) as mucoadhsive polymers to impart mucoadhesion and ethyl cellulose to act as an impermeable backing layer. The formulations were prepared by direct compression and characterized by different parameters such as weight uniformity, content uniformity, thickness, hardness, swelling index, in vitro drug release studies, mucoadhesive strength, and ex vivo permeation study. As compared with the optimized formulation composed of OH—5 mg, CP 934—30 mg, SCMC LV—165 mg, PEG 6000—40 mg, lactose—5 mg, magnesium stearate—1.5 mg, and aspartame—2 mg, which gave the maximum release (88.15%), non-bitter (OH) that form namely ondansetron base and complexed ondansetron was used in order to make the selected formulation acceptable to human. The result of the in vitro release studies and permeation studies through bovine buccal mucosa revealed that complexed ondansetron gave the maximum release and permeation. The stability of drug in the optimized adhesive tablet was tested for 6 h in natural human saliva; both the drug and device were found to be stable in natural human saliva. Thus, buccal adhesive tablet of ondansetron could be an alternative route to bypass the hepatic first-pass metabolism and to improve the bioavailability of (OH).     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.