Immobilization of Pichia pastoris cells containing alcohol oxidase activity

The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran [CSMPI]. Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide [CTAB] and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD[420]. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media

[Extraction and molecular determination of major outer membrane proteins of Brucella abortus S99]

Background: brucellosis is one of the five common bacterial zoonosis caused by a gram negative, non-spore forming, and facultative intracellular bacterial organism belonging to the genus Brucella. Although brucellosis is considered as a health problem for both men and domestic animals in many countries, any licensed human vaccine has not been designed and produced for it yet. To overcome the problem, currently, antigenic determinants of Brucella cell wall e.g. outer membrane proteins [OMPs] and lipopolysaccharide [LPS] are considered as potential candidates to develop subunit vaccines

Materials and Methods: brucella abortus S99 used in the present study is obtained from the standard bacterial collection of Institute Pasteur of Iran. OMPs were extracted by deoxy cholate extraction technique and further purification performed by sequential centrifugation and ultracentrifugation. Protein concentration was determined using the Nano drop ND-10000 spectrophotometer. SDS-polyacrylamide gel electrophoresis [SDS- PAGE] was performed to determine the electrophoretic pattern and the molecular weight of the extracted OMP samples

Results: OMPs concentration of B.abortus S99 has been measured and reported as 6.27 mg/ml. SDS-PAGE analysis indicated one protein band in the range of 36-38 kDa which would be classified as the porins of B.abortus S99

Conclusion: extraction of B.abortus S99 OMPs with the applied method in the present study produced a satisfactory yield of OMPs. These proteins belonging to the second group of OMPs, called porins

[Improved Diagnosis of brucella in human serum samples by double PCR assay]

Brucellae are gram-negative intracellular pathogens which cause zoonotic disease in humans. Clinical manifestations of brucellosis in human are variable and often are non-specific, and the diagnosis requires fast and accurate confirmation. Since the usc of serum instead of whole-blood samples offers several advantages for nucleic acid amplification methods, in this study we developed an improved PCR assay for the rapid and specific laboratory diagnosis of human brucellosis directly from serum specimens. DNA was extracted from 100 microL of serum from 30 patients with acute serologic brucellosis. The PCR reaction was carried out with Specific primers. Second PCR reaction for reamplification of the first reaction products was designed. A 223 bp conserved region on the sequence encoding the 31-KDa immunogenic outer membrane protein which is specific to the genus Brucella [BCSP31] and present in all its biovars was amplified in all serum samples. For confirmation and efficient amplification of the specific target, reamplification of the first PCR products had a sharper banding patterns with high sensitivity and specificity that might be considered as a new useful method for diagnosis of human brucellosis in serum specimens

Safety and immunogenecity of human therapeutic morphine vaccine in morphine addicts

Morphine vaccine is a product of morphine-6- succinate synthesis and its conjugation with albumin serum. Each dose contains 0.5 mg Aluminum hydroxide, 8 mg sodium chloride, 1.12 mg di- sodium hydrogen phosphate, 1.1 mg sodium di- hydrogen phosphate and 50 mg morphine-6-succinate albumin serum. Humoral safety is achieved in morphine addicts following three successive doses within 0-30-60-day intervals. Its immunogenecity brings about withdrawal without the risk of abstinence syndrome. This study was conducted to examine the effect morphine vaccine on morphine addicts. Based on the Ethics protocol of Pasteur Institute of Iran, this clinical trial was conducted on 102 male volunteer addicts [mean age 25 years]. Variables included vaccine dosage and concentration on of antibodies. Volunteers were divided into groups of 30 [experimental] and 4 [placebo]. Adjuant was injected to placebo group addicts; the three experimental groups were given 5, 12, 100 and 600 microg within 0-30-60 days interval through injection to deltoid; all subjects referred for follow-up on the 90[th] day. Blood samples and antibody evaluation was obtained from all three groups in months 5, 7, 9, 11 and 12. A positive correlation was observed between antibody production and vaccine dosage as well as frequency of injection. Anti-morphine antibody was detected after the first injection of 100 microg/ml, 600 microg/ml and second injection of 12.5 microg/ml doses. Antibody levels reached the peak by three months and did not decline to the baseline after one year. The vaccine was well tolerated and lacked the reverse and unfavorable effects, characteristic of vaccines and drugs. On day 90, humoral immunity was created in all patients

Surveying Desferal production increment by inducing mutation in Streptomyces griseoflavus

Desferal or Desferrioxamine B mesylate is an iron chealator drug. This medicine decreases the iron overload in the thalassemia patients who have been blood transfused; the excess of iron is excreted through bile or urine. Novartis is the sole company which produces desferrioxamine B mesylate in the world and our country is importer of such drug. Thus we tried to increase Desferal production by inducing mutation in Streptomyces griseoflavus. This is an applied research carried out at pilot level. The organism is a Gram-positive bacterium that was supplied in lyophilized by Persian Type culture Collection, Iranian Research Organization for Science and Technology [IROST], Tehran, Iran bearing the code no. PTCC1130, which was cultured on Des4 medium. The organism was mutated by UV irradiation hence selective techniques and markers were employed to distinguish marked strains from parent S. griseoflavus. When the mutated organisms were selected according to their characteristics and used to fuse their protoplasts in order to obtain high yield desferrioxamine producing recombinant Streptomyces griseoflavus. The varied parameters were bacterial growth rate and desferal concentration in the culture broth. Our study showed that the rate of desferal production in mutant's strains called C7031 and S7011 and fusants srains called FP10 and FP9 was higher than wiled type Streptomyces griseoflavus. The increment in production of desferrioxamine was found to be 68% in FP9 and 81% in FP10 fusants. The mutation and protoplasts fusion of Streptomyces griseoflavus caused increment in production of desferrioxamine. The infrared spectrum, thin layer chromatogram of desferrioxamine extracted from culture broth was similar to that of standard desferrioxamine [Novartis] from the point of molecular identity

[Assessing sensitivity, specificity and accuracy of a modified rapid urease kit for clinical diagnosis of Helicobacter pylori]

Rapid urease test is increasingly used as a cheap and quick procedure with high specificity for clinical detection of Helicobacter pylori worldwide. To evaluate the functionality of a rapid urease kit manufactured by Pasteur institute of Iran in 2002, with some modifications compared with other common kits. During a clinical trial in Baghiatallah hospital, Tehran, the sensitivity, specificity and accuracy of the kit was investigated. This was an experimental study in which 91 patients suspected of having H pylori infection were examined. Following collection of history and clinical symptoms, the endoscopic signs of all patients were also recorded. Each endoscopic specimen was carefully examined in three ways with no previous knowledge of operator as follows: 1. Detection of urease test using modified rapid urease kit. 2. Detection of urease test using certified commercial kit. 3. Pathologic examination as the gold standard method. Regarding the results of present study, employing modified rapid urease kit produced positive results on 78.8% of pathologically positive samples. When negative pathologic samples were tested with modified kit, 89.7 % of samples found to be negative. The differences observed between the results of two tests were statistically significant [p=0.001]. Using modified rapid urease kit also produced a false positive rate of 4.4%. Based on our data, it was shown that the sensitivity, specificity and accuracy of the modified kit were 78.8%, 89.7% and of 83.5%, respectively