Iranian Journal of Biotechnology
Year:2003 | Volume:1 | Issue:4|Papers Count:8

The advantages of immobilized enzyme over its soluble counterpart arise from their improved stability and easy separation from the reaction media, leading to decrease in production cost. Immobilization methods range from adsorption onto matrices, entrapment, cross-linking and covalent bonding to prefabricated carriers or activated supports. Changes in kinetic properties of immobilized enzyme can produce substrate or pH gradient, which reduce the reaction rates and finally product yields.

Production and purification of human growth hormone using a simple method was studied in two recombinant Escherichia coli, D7-5 and C27-2 strains. The r-hGH was expressed in the form of inclusion body in a batch fermentation process and purified to 99% purity using a procedure based on acid precipitation of the host derived proteins and other impurities. The effect of the pH and host strain on purification of the r-hGH and efficiency of the procedure were evaluated. It was found that the optimum pH for precipitation of the host derived proteins was 4.9. The procedure was suitable for r-hGH purification from D7-5 stain but not from the other strain C27-2. The purity of > 99% and recovery of about 40% were obtained as shown by SDS-PAGE and Western blot analysis. The purified r-hGH was biologically active as judged by receptor assay with very low endotoxin content which could be suitable for therapeutic applications. This simple and cost effective production process could be useful for large scale production of recombinant hGH from specific strains.

The effectiveness of the first Iranian made gonadotropin releasing hormone analogue, [D-Ala6 des-Gly10] GnRH ethylamide, alone or in combination with domperidone, a dopamine antagonist on spawning rate, latency period, working fecundity and embryo viability in common carp, Cyprinus carpio, was investigated. Fifty two fish were divided into 5 groups and treated intrapretoneally as follows: 3 mg/Kg b.w. of carp pituitary extract (C.P.E.) as a positive control, GnRHa alone, 10 µg/Kg b.w.. or in combination with domperidone, 5 mg/Kg b.w. in a single or double injections 7h apart. A group was treated with propylene glycol 0.2 ml/Kg b.w. alone and considered as control. No female ovulated in groups receiving either propylene glycol or 10 µg/Kg b.w. of GnRHa alone. The spawning rate was higher in female GnRHa+domperidone (10 µg/Kg b.w..+5 mg/Kg) in double injections (11 out of 12) as compared to fish which injected either with C.P.E (7 out of 16) or GnRHa + domperidone in a single injection (3 out of 12)(P<0.05). The mean working fecundity, was significantly higher for fish receiving GnRHa+domperidone in single (126214 ± 24315) or double injections (145600 ± 27113) compared to C.P.E treated group (52435 ± 1224) (P<0.05). There were no significant differences for latency period or embryo viability among the groups.

In the budding yeast of Saccharomyces cerevisiae the tandem repeated of rDNA genes are located on chromosome XII, which is in the nucleolus. There are different types of proteins in the nucleoluskeleton, silencing proteins have got important role in nucleolus. It is shown that meiotic recombination between nonsister chromatids in the rDNA genes are strongly suppressed, and suggested that silencing proteins such as SIR2 are involved in silencing state. It is also shown that nucleolus shows some changes during ageing process. It is claimed that intrachromosomal recombination within the rDNA repeated sequences; producing 3-µm rDNA circles are accumulated with old cell. This study looked at the rDNA breakage in two different types of strain ORD 1181 according to their age. The fine analysis of the rDNA array was performed using restriction endonuclease enzymes, which do not cleave within the rDNA array. The results suggest that there are only meiotic hot regions for chromosome breakage in the old cells within the rDNA array.

In an extensive numerical phenetic survey, a number of blue, red and gray spored streptomycete strains were grouped together as Streptomyces cyaneus species-group, a taxon which encompasses strains known to produce antitumor antibiotics, notably anthracyclines. In the present investigation these and related streptomycetes were the subject of morphological and 16S rRNA sequencing studies designed to clarify their taxonomic relationships. It is evident from these results that the Streptomyces cyaneus species-group encompasses misclassified strains and members of several distinct species. However, some of the red-spored strains (e.g. Streptomyces janthinus ISP 5206T, Streptomyces roseoviolaceus ISP 5277T and Streptomyces violatus ISP 5209T) formed a distinct clade. In contrast, most of the blue and gray-spored strains showed much less sequence homology between and with one another and were scattered throughout the 16S rRNA Streptomyces tree. The improved classification of this group of streptomycetes provides an essential basis for establishing their industrial and economical significance.

The present study was conducted to identify the organogenetic potential of cotyledon sections from different genotypes of sunflower. Seeds were surface sterilized and germinated on hormone free half strength MS basal medium. Cotyledons from 2-dayold seedling were split in half and cultivated on MS medium supplemented with 4.4 µM BAP and 5.4 µM NAA. The experiment was designed in a randomized complete block with 3 replications. There were differences among the genotypes and cotyledon sections for all studied organogenesis parameters. Inbred line CMS60/52 and proximal explants presented the highest values for the percentage of explants producing shoots, 57.1% and 58.5%, respectively. CMS60/52 × proximal explants, interactions showed the highest values for average number of shoots per explants plated as well (13.3) as regenerant explant (21.3 shoots) respectively. Shoot organogenesis was optimized on proximal explants.

A rapid, sensitive, specific and high through-put enzyme-linked immunosorbant assay (ELISA) method for determination of morphine in urine samples using penicillinase as label enzyme has been developed. No extraction or chromatography was included in this assay procedure. Immunoglobulin (Ig) purified polyclonal anti-bodies against a C6-hemisuccinate derivative of morphine (M-C6-HS) conjugated to bovine serum albumin (BSA) was coated onto the wells of microtiter plate. A morphine-C3-hemisuccinate (M-C3- HS) was also prepared and the two derivatives were conjugated to penicillinase (M-C6-HS-P and M-C3-HSP). The heterologous combination of antibody prepared against M-C6-HS-BSA and enzyme conjugate prepared for M-C3-HS-P showed better properties in term of sensitivity, reproducibility and slope of standard curve. The assay was sensitive from 20 pg/ml and detected up to 100 ng/ml of morphine in urine samples. The affinity of antibody in homologous assay was found to be 6.6×1010 l/mol and for heterologous assay was 3.2 ×1012 l/mol. The assay was completed within 4 h. The homologous assays performed under different conditions of coating, concentrations, duration, pH, etc. did not end up with a suitable standard curve. Hence it seems that the ability of morphine to displace the hapten enzyme conjugate dependds on the position of the enzyme coupled to the hapten molecule. This ELISA techniqu showed 100% correlation with immunochromatography (IC) and 90% percent correlation with latex agglutination inhibition (LAI) test in the results obtain with urine samples declared positive by authorities. ELISA also showed approximately 90% correlation with LAI-negative urine samples

Cell culture technique has been used for detection and confirmation of many different viruses in clinical samples. Although, new diagnostic methods have been developed for viral infections, traditional cell culture technique is still regarded as the “gold standard” for several infectious agents such as human cytomegalovirus (HCMV). In the present study, a new human fetal foreskin fibroblast (HFFF)-derived cell line was obtained from a normal Iranian male fetus at the end of first trimester at National Cell Bank of Iran (NCBI) and its ability for HCMV growth was investigated. Thirteen treated urine samples from renal transplant recipients were inoculated onto HFFF monolayer. HCMV growth was monitored and confirmed by typical CMV cytopathic effect (CPE), histological staining, immunological staining and polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method and sequencing. All of the abovementioned detection methods were confirmed that new HFFF-derived cell line is a sensitive line for growth of HCMV. This cell line has been named HFFF-PI6 and is available with accession number NCBI C170 at NCBI of Pasteur Institute of Iran.