Research in Pharmaceutical Sciences
Volume:4 Issue: 1, Apr 2009

Tenecteplase is a variant of tissue plasminogen activator (t-PA) which has better pharmacokinetic properties and more selective thrombolytic activity. In the present study, we describe a rapid method to introduce three sets of mutation into defined positions in t-PA cDNA by a site-directed mutagenesis based on a megaprimer PCR approach to produce tenecteplase coding sequence where amino acids at positions 103, 117 and 296-299 in polypeptide sequence were modified. This sequence was cloned in pTZ57R by T/A cloning method to introduce suitable restriction sites at both ends of the amplified fragment. Vectors containing tenecteplase cDNA were propagated in One Shot TOP10 chemically competent E. coli and positive clones were selected by blue/white screening method. After being isolated and purified, the recombinant plasmids were digested by suitable restriction enzymes. Acquired tenecteplase coding sequence was subcloned into a tissue specific expression vector. Upon expression, it is expected that tenecteplase will be expressed exclusively in lactating mammary glands during milk production in transgenic animal. Finally, the recombinant vector was isolated and verified by restriction digestion and sequence analysis to confirm that the tenecteplase coding sequence has been inserted in the proper orientation.

In the present study, a simple and sensitive extractive spectrophotometric method is described for determination of buspirone. The method is based on the reaction of buspirone and bromocresol green. The ion-pair complex was quantitatively extracted into chloroform at pH 2.3 followed by spectrophotometric determination at 415 nm. The complex was stable up to 2 days and obeyed Beer''s law over the concentration ranges of 1.5-6 µg/ml. No significant interference was observed from the excipients, coloring and flavoring agents commonly used in the buspirone pharmaceutical preparations. The proposed method has been applied successfully for determination of buspirone in commercial pharmaceutical preparations.

Coumarins are bioactive secondary metabolites and could be found in the fruits of Umbelliferae. Prangos asperula Boiss. is an Iranian native plant which is found wild in many regions of Iran. Osthol, a prenylated coumarin, was isolated from the hexane extract of the fruits of P. asperula and its structure was elucidated using 1HNMR, 13CNMR, IR and MS spectra. The essential oil of the aerial parts of the plant obtained by hydrodistillation was also investigated by GC-MS. Forty-seven constituents have been identified of which 2,3,6-trimethyl benzaldehyde (18.4%), d-3-carene (18.0%) and α-pinene (17.4%) were the main constituents of the oil.

The present study was aimed to isolate and characterize the lipolytic enzyme producing bacteria from soil samples of regions around Zayande-rood river of Isfahan, Iran. Soil samples were collected from 15 cm depth of soil surface. Based on morphology, distinct colonies were isolated and purified through streak culture on to standard agar plates. Isolated colonies were examined for lipase activity using egg-yolk agar medium. Total of 15 isolates developed clear zones around their growth area which were considered as lipase positive. Preliminary identification of lipolytic active isolates revealed a gram-positive, rod-shaped, endospore-forming and catalase positive bacteria, characteristics indicative of the genus bacillus. The gene coding for an extracellular lipase was cloned using PCR techniques. The gene was identified to be 639 bp in length and encoded a peptide of 212 amino acids with calculated molecular mass of 19353 Da, and pI 9.28. The DNA sequence and deduced amino acid sequence of the hypothetical lipase showed striking similarities to lipases from B. subtilis strains.

Angiotensin-II is a multifunctional hormone that regulates blood pressure, plasma volume, neuronal functions, electrolyte balance, thrust and various other vital mechanisms. It acts through its receptors AT1 and AT2. The involvement of angiotensin-II and its receptors in cognition is still ambiguous. Therefore, the present study was designed to investigate the effect of angiotensin-II (2 μg/3 µl, i.c.v.), losartan (AT1 blocker) (20 mg/kg, i.p.) and PD123177 (AT2 blocker) (20 mg/kg, i.p.) on scopolamine (3 mg/kg, i.p), sodium nitrite (75 mg/kg, i.p.) and BN52021 (15 mg/kg, i.p.) induced amnesia in mice using water maze test. All the agents were administered 30 min prior to first acquisition trial for 4 consecutive days and on the 5th day during the retrieval trial. The study indicated that losartan significantly reversed the scopolamine and sodium nitrite induced anterograde amnesia. On the other hand losartan failed to produce any significant effect on sodium nitrite and BN52021 induced retrograde amnesia. Angiotensin-II and PD123177 did not exhibit any significant effect on scopolamine, sodium nitrite and BN52021 induced amnesia. The findings suggest that anterograde amnesia of scopolamine and sodium nitrite may be mediated through AT1 receptor subtype only. These receptors are not involved in retrograde amnesia of sodium nitrite and BN52021. The finding also indicates that the brain structures involved in learning and memory are insensitive to exogenous angiotensin-II and PD123177 or have very less density of angiotensin receptors particularly AT2 subtype.

The aim of this study was to clone the serine alkaline protease-encoding gene from Bacillus subtilis 168. This protease, which can have many applications especially in detergent, may be industrially an important enzyme. For the amplification of the gene, PCR was performed with a pair of primers specifically designed for this purpose. Electrophoresis of the PCR product showed the expected band of 1329 bp. Restriction analysis also confirmed the integrity of the PCR product. After ligation of amplified gene into pTZ57R by the method of TA cloning, digestion with appropriate restriction enzymes confirmed the integrity of the cloned gene. Successful cloning of the protease gene from B. Subtilis could pave the way for the expression studies in a suitable host.

A sensitive, accurate and rapid reverse phase HPLC method was described to quantitate levels of acyclovir in human plasma. The drug, internal standard (metronidazole) and phosphate buffer (0.05 M) were added to serum samples and vortexed for 30 sec. A mixture of isopropyl alcohol:dichloromethane (60:40) was then added and vortexed for 3 min. Samples were centrifuged and the supernatant layer was separated, evaporated to dryness under nitrogen gas stream, reconstituted in mobile phase and, an aliquot of 50 μl was analyzed on a μ-bondapack C18 (250 × 3.9 mm) column, with 3% acetonitrile in deionised water and 0.5% orthophosphoric acid, (pH 2.5) at 254 nm. The standard curve covering 100-1500 ng/ml concentration range, was linear, relative errors were within 0.79 to 17.4% and the CV% ranged from 3.81 to 18.2. The limits of quantitation and detection of the method were 100 ng/ml and 25 ng/ml, respectively. The method was suitable for bioavailability and pharmacokinetic studies of acyclovir in humans and applied in a randomized, two-way cross over bioequivalence study of two different acyclovir preparations with twelve subjects and with a one-week washout period.