IRANIAN JOURNAL OF BIOLOGY
Year:2009 | Volume:22 | Issue:1|Papers Count:23

Penicillin acylase (penicillin amidase, PAC; EC 3.5.1.11) is the key Enzyme used in the industrial production of b-lactam antibiotics. This enzyme cleaves Penicillin G to 6- amino penicillanic acid (6-APA) and PAA by hydrolysis of acyle side chain. It can process this reaction in both directions (hydrolysis and synthesis). Here, we sought to clone and express the penicillin G acylase gene from A. viscosus in Escherichia coli, to improve expression level. Following PCR amplification of PAC gene from genome of A. viscosus, PAC gene was cloned in pET26(b) expression vector in frame with DNA sequence coding for 6xHis tag presented at 3' of MCS of the vector. After sequencing and confirmation of gene identity with published sequence (L04471), positive clones were screened based on differential response of Serratia marcescens (ATCC27177) to penicillin G and 6-APA (resistance to penicillin G and susceptibility to 6-APA). Furthermore, SDS-PAGE and western blotting with anti.His.HRP. Conjugate antibodies confirmed the autocatalytic processing in cytoplasm to produce the active enzyme. Our results shows the ability of E.coli (BL21) strain in expression of active pac gene from a gram positive bacterium in the cytoplasm.. Furthermore, our results show the benefit of T7 promoter and IPTG induction in obtaining high expression levels in E.coli for penicillin G acylase from a gram positive bacterium.

In the present study, microorganisms were isolated by screening methods from Ferula galbanum Boiss, galbanum gum and surrounding terpene soaked soils which are nature source of oily ethers. Twenty four microorganisms (15 fungi, 9 bacteria) were isolated. Microorganisms were tested for their ability to produce a-pinene in the present b-pinene. The bio transformation medium involved, 100ml buffer phosphate (Na2HPO4, NaH2PO4), pH 6, 100 ml b-pinene, and 19 biomass, 37°C, 150rpm and 22 h. Biotransformation products were extracted with n-hexan and analyzed by gas chromatography. One bacterial isolated capable of forming b-pinene to a-pinene. The growing conditions i.e. carbon source and nitrogen source were optimized. The maximum cell density were 33.59 g l-1 and 40.44 gl-1 in presence of 30 gl-1 glycerin as carbon source and 7 gl-1 urea as nitrogen source, respectively. The biotransformation rate was improved under optimal conditions. The maximum a-pinene were formed as 0.133% and 0.205 % with 20 gl-1 glycerin and 7 gl-1  urea were applied as carbon and nitrogen sources , respectively.

Aran-Bidgol salt lake is one of the lakes in central desert zone of Iran and is largest playa in Iran. This lake has shape as like a triangle that top it is towards north. Base length this triangle is 35 Km and high it is 38 Km. Screening bacteria from different points Aran-Bidgol salt lake, led to the isolation of 61 Gram-positive halophilic bacteria and 22 Gram-negative bacteria able to produce different hydrolytic enzymes. These strains are able to grow optimally in media with 5-15 % salts, 35-37°C temperature and pH 7.2. Strains No. 32, 20, 27, 40, 40, 9, 11, 24, 16 and 20 were produced amylase, protease, lipase, DNase, inulinase, xylanase, carboxy methyl cellulase, pectinase, pullulanase and chitinase. It is interesting that combined hydrolytic activities have been detected in some strains. DNase, indulines were the most enzymes produced in Grampositive bacilli and Gram- negative bacteria, lipase and Gram-positive cocci, pullulanase, carboxy methyl cellulase. Among these isolates, several strains with the ability to produce different valuable enzymes were selected, including AMB1(8 enzymes); AMB7, AMBI0, AMB12(7 enzymes); AMB5, AMB6, AMB11, AMB13(6 enzymes); AMB2, AMB3, AMB8, AMB9(5 enzymes); AMB4, AMGl(4 enzymes); AMCl, AMC2(3 enzymes); AMC3, AMC8, AMCI7, AMG2 (2 enzymes); AMG3(1 enzymes). Selected strains, after more accurate physiological and biochemical studies were identified regarding phylogeny and molecular characteristics using 16S rRNA technique. Sequencing 16SrRNA was performed to complete the identification process, which Gram-positive bacteria were belong to Halobacillus, Thalassobacillus, Bacillus, Salinicoccus and Gram-negative bacteria were belong to ldiomarina, Salicola and Halomonas.

D-xylose, is the second most abundant sugar in nature and it is one of the main constituents of plants. Hence, ethanol production from D-xylose has an ignorable importance in the world future energy trends. In this research, natural environments and materials such as compost and decaying body of plants and agricultural wastes, were investigated for finding xylose fermentating yeasts, which can produce ethanol using D-xylose. First, one gram of different samples were cultured in broth culture media comprising xylose as the only energy source and yeast extract and after 24 hours 0.1 ml of brothes were transferred on solid medium containing the same elements. D-xylose utilizing yeasts were examined for their ability to ferment D-xylose using their culture in mild agitation. From 61 isolates, 5 isolates could utilize D-xylose and produce less than 1 g/l ethanol from 20 g/l D-xylose and 7 isolates could produce more than 1 g/l ethanol from 20 g/l D-xylose. These later isolates were Candida boidinii, Candida paludigena, three isolates of Kluyveromyces marxianus and two isolates of Auerobasidium polullans. Among these isolates, Candida boidinii with 5.8 g/l ethanol from 20 g/l D-xylose was the best ethanol producer. As a result of this study, 100 rpm agitation, 5 g/l yeast extract, 60-84 hours incubation time and pH=4.5 were the best environmental condition for the maximum production.

Filamentous fungi such as Trichoderma spp. Produce a variety of chitinase enzymes that degrade chitin and play an important role in biological control of fungal diseases. Chitinases are hydrolysis enzymes which degrade chitin, a linear biopolymer of Nacetylglucosamine, into its component residues by breaking the b-1,4 glycosidic bonds. In this research Trichoderma atroviride PTCC5220 an over producer of chitinase enzyme among 30 Trichoderma sp. isolates used for study of chit42 gene. Two specific primers (CUM1/CDM2) and genomic DNA from T atroviride were used for chit42 amplification. The amplified DNA fragment (about 1.6 kb) was analyzed and confirmed by restriction pattern using XhoI enzyme.The chit42 DNA fragment was cloned into pUC19 and designated as pMJH1. Comparison of the cloned fragment with the cDNA sequence indicated that this gene contains three short introns (58bp, 62bp and 70 bp long) and also an open reading frame coding for a protein of 421 amino acids. Alignment of the amino acid sequence with other reported Chit42 sequence from Trichoderma sp. (T. aureoviride, T viride, T harzianum, T. longibrachiatum and T koningii) showed 100%, 98%, 98%, 97% and 96% identity, respectively. 

Mentha piperita is the important species of genus Mentha and it's essential oil is desired. In this research amount of essential oil, menthone reductase activity and antibacterial activity of essential oil were investigated in two stage of growth, 2 and 4 months after sowing. The results showed that essential oil composition of M piperita in two harvests was different. In first harvest, the main constituents of the oil were Menthone, Menthol, Menthofuran, Pulegone, 1,8- Cineole and menthyl acetate respectively whereas the main constituents of the oil in second harvest were Menthol, Menthone, Neo-Menthone, 1,8- Cineole and Menthyl acetate respectively. Menthone reductase activity was significantly greater in the leaves that were harvested 2 months after sowing compared to the 4 months. The results of this research showed that the amount of Menthol was increased accompanying with increase of the enzyme activity. Antibacterial activity of the oil were tested against eleven Gram-(+) or Gram-(-) bacteria. It was found that the oil of first harvest had antibacterial activity against all bacteria and was more active than the essential oil of second harvest.

Different species of Bacillus and Pseudomonads are among the most important bacterial antagonists that are capable of controlling phytopathogenic fungi and bacteria. In the green house experiments, 5 bacterial isolates belonged to Bacillus (Sch-5, Sch-3, Ssh- 4,Ssh-3) and Pseudomonads Ckk-1 which showed highest effectiveness in controling of Rhizoctonia solani (isolates RsK and RsA) were selected. In experiment for investigation of Antibiotic production percent inhibiton of mycelial growth of R solani by bacterial isolates was determined. This experiment carried out for RsK isolate in 3 treatments(2 bacterial isolates and control) with 3 replications and for RsA in 5 treatments (include 4 bacterial isolates and control) with 3 replications. Growth of RsA on NAG was determined 5 days later. The results showed that Sch-3, Ssh-3, Ckk-1.Ssh-4 inhibitory effect against RsA were 58/8%, 62/2%, 74/4%, 83/3% respectively. Isolates Sch-3' and Sch-5 inhibitory effect were 68/8% and 60% against RsA respectively. In another experiment, to study volatile metabolites produtions in 3 treatments ( 2 bacterial isolates and control) with 3 replications for RsK and in 5 treatments (4 bacterial isolates and control) with 3 replications for RsA. The results indicated that fungal isolate RsK growth was about 8mm against bacterial isolates Sch-5, Sch-3 and fugal isolate RsK against Ckk-1, Ssh-4, Sch-3, Ssh-3 was 8, 8, 10, 12mm respectively. There were significant differences among control and other treatments. The overall results indicated that all bacterial isolates could produce volatile and non-volatile metabolites and Ckk-1 isolate of Pseudomonas was capable of siderophore and hydrogen cyanide production which are their most important mode of action against plant pathogenic microorganisms.

One of the new approaches for targeted therapy of human disease like cancer is the use of molecules so called immunotoxins. Immunotoxins, comprised of both the cell targeting and the cell killing moieties. In the last few years a new approach for targeted therapy of human disease has been developed using cytotoxic molecules that are produced by gene fusion techniques. This class of molecules termed chimeric proteins comprises both the cell targeting and the cell killing moieties. In this investigation, the already constructed chimeric protein containing catalytic domain of Shiga-like toxin (Al) fused to human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and cloned in E. coli was expressed by arabinose induction. The expressed protein extracted and then was analyzed for its cytotoxic activity on the cells bearing GM-CSF receptor. To elucidate the apoptotic activity of hybrid protein, the expressed protein was added to the cells and the apoptotic activity was measured by cellular DNA fragmentation ELISA kit (Roche). The results indicated that the chimeric protein was cytotoxic for the cells bearing GM-CSF receptor and it was shown that different concentration of the chimeric protein induced apoptosis on the cells measured by the kit. The overall results indicated that cytotoxic activity of the chimeric protein is specific on the cells and it can also induce apoptosis.

In this research effects of 25, 35, 45 PPt. salt concentrations & different intensities of luminance 2000, 3500, 6000 1ux have been used to purification and isolation of two unicellular algae Nannochloropsis sp. and Isochrysi sp. in a mixed medium. Walne medium culture containing B12 and B1 vitamins in pH 8.The growth of algae was studied during a 7 days period. Results showed that Nannochloropsis oculata has the highest growth in 25 PPt salt concentration and 6000Lux. luminance. While Isochrysis galbanum has the least growth under these conditions. So it is concluded that we can isolate and purify these two algae and by 100%purification by this way. We suggest the method is a suitable method to purify unicellular algae to have some special researches on them.

Having a diversity of necessary amino acids and fatty acids (W6 & W3) Tubifex worm (Tubifex tubifex) is a valuable food source for many ornamental fish species as well as larva of other fishes. Due to the risk of transferring pathogens, collection of these worms from natural environment is not recommended. We investigated the influence of culture substrate on biomass production of worms in a completely randomized design with two treatments (substrates) each with four replicates. The substrate treatments were organic fertilizer (cow dung) + sand with a height of 3 cm (treatment 1) and minced lettuce + sand with the same height (treatment 2). (The proportion of cow dung or lettuce to sand was 4:1 in all the treatments and the diameter of sand granules was 0.25 to 0.40 mm). The experimental units were small aquariums (length = 30 cm; width = 25 cm; height = 20 cm) adjusted to 100 ml/minute flow rate of water after preparing substrate treatments. Treatments 1 and 2 had 20.5 and 46.6 times increase respectively in the number of Tubifex worms at the end of a 45-days period of culture. The mean number of produced Tubifex worms was significantly different between the two treatments (P< 0.001).

Production and rearing of all female triploid populations of rainbow trout Oncorhynchus mykiss, has great importance in aquaculture. Decrease or suppression of ovary development of all female triploid populations results in increasing growth and flesh quality. Hence, in this research, ovary development of all female triploid rainbow trout was investigated and analyzed by classic histology in compare to all female diploid population in the second year of culture. One year investigation results during 6 stages of sampling showed that germ cells of triploid fish ovary developed at most up to 2a stage and then their development was suppressed until the end of sampling period but the ovary of diploid fish normally developed and reached to 4b and 5 stages until the end of sampling. In conclusion it can be stated that triploidy induction on all female populations of rainbow trout results in considerable suppression of ovary development in these fish and consequently increasing somatic growth in compare to diploid counterparts.

In this study, the general morphology, ultrastructure of the cells and the localization of Na+- K+ ATPase were examined in the antennal glands of Astacus leptodactylus. Each gland is composed of four units, including the coelomosac, the labyrinth, the tubule and a voluminous bladder, linked by a short duct to the urinary pore. Ultrastructural observations have shown that the cells of coelomosac possess podocytic feature. These cells have Golgy complex systems, vacuoles, endocytotic vesicles and they possess distinctive pedicels. The labyrinth cells (cuboidal and columnar cells) present basal membrane infoldings, apical vesicles, apical microvilli and apical cytoplasmic extrusions. The tubule is consist of two sub-unit (proximal and distal tubules) and the cells present in common basal membrane infoldings associated with mitochondria, and cytoplasm including numerous vacuoles and mitochondria. These cells have not apical microvilli and they also present distinctive characters in each sub-unit. Na+-K+ ATPase was detected through immunofluorescene in the tubule and bladder cells with an increasing concentration. A weak fluorescence was also shown in labyrinth and no immunofluorescence was detected in the coelomosac. The cells of the tubules and the bladder present morphological and enzymatic feature of ionocytes. Thus, the antennal glands of the A. leptodactylus possess active ion exchanges capabilities and participles in osmoregulation.

Glucagon-like peptid-1 (GLP-1) and Oxyntomodulin is potent hormone released fro gut and have important actions on peripheral tissues and CNS including appetite control. GLP-1 nerve fibers and terminals are widely distributed throughout the brain, with the highest density occurring in the hypothalamus, thalamus and septal regions. The aim of the present study was to investigate the effect of central oxyntomodulin (OXM) injection on expression of GLP-1R mRNA in the brainstem of chicks. RT-PCR results revealed that expression of GLP-1R mRNA was decreased 30 and 90 min after injection of OXM. The present finding shows a negative regulatory effect of OXM on GLP-1R mRNA expression in the brainstem of chicks.

The aim of this study was to examine the effect of water pH changes on certain haematological parameters of fingerlings of common carp (Cyprinus carpio L.), in water with different pH (acidic & alkaline). Fingerlings of common carp subjected to acidic (pH 5.5, 6.5) and alkaline (pH 8.0, 8.5 and 9.0) water for 21 days. Control groups were maintained at neutral pH. In this study changes in shape, size and population of blood cell also hemoglobin concentration and blood glucose of fishes were determined as stress indexes. Result showed that exposure to both acidic and alkaline water exerted stress in fish and considerably affected the haematology of fingerlings of common carp. The changes in hematological parameters of common carp fingerlings indicated that the change in water pH might have caused the ion regulatory and acid-base disturbances originating at the gill leading to the altered internal pH, electrolyte and osmotic balances that imply an increase in energy consumption to restore homeostasis instead of other physiological functions and weight gain and growth.

Poplars are species, because of rapid growth, possibility of dense plantation, annual high growth and short-rotation harvest, that recently it was planted in the different places of Iran. Thus colons identification prosperous populous and study growth characteristics in the primary stage growth this species value which have nowadays main proportion on woody production in the industry country, they are specific important. In this study in order to investigation of the effects growth traits and morphological populus species on primary stage growth, ramets were collection in the three region of the northern of Lorestan province (Aleshtar city, Khoramabad and Nourabad) plantation in nylon vases on nursery. In this study measured and recorded factors including: length leaf, width leaf, length to width proportion, coefficient form, perimeter and area, length leaf stalk, number of leaf, diameter of collar and height in the initial and second stage of sapling. Results showed that case study factors of saplings are significant different (exception of length to width proportion and coefficient form) in the three region with probability (P<.001). Under study factors have good function as morphological marker in identification of the clones' different environmental areas. It was observant that region Nourabad different in the region more than each other areas. That is condition for the reproduction, rowing and development species in those regions very important.

Alternaria fungi cause two different diseases on citrus in Mazandaran province, North of Iran: Alternaria brown spot of tangerines and their hybrids, and Alternaria black rot of the Navel orange fruits. In this study, infected tissues collected from infected leaves, fruits and young stems of the plants. The tissues were cuted to (2x2 mm) pieces and fixed over night at gluteraldehyde and phosphate buffer, post fixation was done with osmium tetroxide, after lyophilization, the specimens were coated with gold and then studied using a SEM (LEO 435). Multiple germ-tubes developed randomly from each conidium and grew in any direction across the leaf and fruit skin surfaces. The hyphal penetration in the plant surface, whether directly through the epidermis or via stomata occured with or without the appressoria formation. The present study showed that occurrence of the infection was through stomata and direct penetration. Hyphal penetration continued through of the sub stomata cavity, and then some of hyphal branches grew in the intercellular space of mesophyll tissue. The hyphal products, specially toxins (HST and NHST), caused cell and cell wall damages. None of the hyphal branches were penetrated in to oil glands of the leaves and specially of the fruits. Based on the observations, the oil gland componements have had inhibitor roles on the hyphal penetration and development.

The effect of toxic concentrations of Ni and Zn (up to 100mm on growth and metal accumulation was studied in some species from flora of Azerbaijan (Northwest of Iran) in a hydroponic experiment. Plant species including Cichorium intybus, Secale montanum, Dactylis glomerata, Salvia sclarea, Alyssum strigosum and Silene compacta are either from important elements of heavy-metal rich soils of Azerbaijan or are excluded from these areas. Growth data of shoot and root showed that Secale montanum is an extreme tolerant species to toxic concentrations of Ni, but Cichorium intybus and Silene compacta are highly susceptible species with growth inhibition of up to 90%. Shoot concentration of Ni in Alyssum strigosum and Silene compacta reached up to 2 mg g-1 DW being lower than the standard value of Ni-hyperaccumulation, but indicating a high accumulating potential for Ni in these species. Growth of Secale montanum and Salvia sclarea was not inhibited by Zn concentration of 100 mM in the medium, in contrast Silene compacta was the most susceptible species for Zn with 80% reduction of shoot growth. Similar with Ni, Alyssum strigosum and Silene compacta showed the highest accumulation of Zn in leaves (up to 1.1-1.7 mg g-1 DW), however, this amount was much lower than critical value for Zn hyperaccumulators (10 mg g-1 DW). In this work, no relationship was found between growth response of plants and heavy metal accumulation in leaves, so that, exclusion and inclusion were observed in both tolerant and susceptible species to Ni and Zn. 

Chalous lowland (Khanikan) forest is located at South of Nowshahr city (with 2807 hectare area). In order to investigate factors of diversity indices sixty plots (400 m2 area each) were taken by a systematic random sampling method. Species - area curve was used for determining plots area. Complete enumeration was used to estimate characteristics of forest stands (growing stock, basal area and diameter at breast height) in each plots. In order to analyze of diversity indices were used Simpson, Shannon wiener, and McIntosh, Hill (N1) and Hill (N2) indices. Results of this research showed that there are positive liner regression between Simpson, negative liner regression between McIntosh, Shannon wiener, Hill (N1) and Hill (N2) indices with respect to characteristics of forest stands. Results indicated that Simpson index have showed the highest correlation related to growing stock (r = 0.71) and diameter at breast height (r = 0.69). Hill (N1) index have showed the highest correlation related to basal area (r = 0.66).