Abstract: Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.
Abstract: T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 1010 pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.
Abstract: The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.
Abstract: Millets as a new source of plant protein were not used in food applications due to its poor functional properties. In this study, the effect of high intensity ultrasound (frequency: 20 kHz, with contentious flow) (US) in 100% amplitude for varying times (5, 12.5, and 20 min) on solubility, emulsifying activity index (EAI), emulsion stability (ES), foaming capacity (FC), and foaming stability (FS) of millet protein concentrate (MPC) were evaluated. In addition, the structural properties of best treatments such as molecular weight and surface charge were compared with the control sample to prove the US effect. The US treatments significantly (P
Abstract: The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.
Abstract: Ambrosia trifida L. is designated as invasive alien
species by the Act on the Conservation and Use of Biodiversity by the
Ministry of Environment, Korea. The purpose of present paper was to
investigate the inhibitory effects of aqueous extracts of A.trifida on the
development of root hairs of Triticum aestivum L., and Allium
tuberosum Rottler ex Spreng and the electrophoretic protein patterns of
their radicles. The development of root hairs was inhibited by
increasing of aqueous extract concentrations. Through SDS-PAGE,
the electrophoretic protein bands of extracted proteins from their
radicles were appeared in controls, but protein bands of specific
molecular weight disappeared or weakened in treatments. In
conclusion, inhibitory effects of A. trifida made two receptor species
changed morphologically, and at the molecular level in early growth
stage.
Abstract: Collagen was isolated from chicken feet by using papain and pepsin enzymes in acetic acid solution at 4°C for 24h with a yield of 18.16% and 22.94% by dry weight, respectively. Chemical composition and characteristics of chicken feet collagen such as amino acid composition, SDS-PAGE patterns, FTIR spectra and thermal properties were evaluated. The chicken feet collagen is rich in the amino acids glycine, glutamic acid, proline and hydroxyproline. Electrophoresis pattern demonstrated two distinct α-chains (α1 and α2) and β chain, indicating that type I collagen is a major component of chicken feet collagen. The thermal stability of collagen isolated by papain and pepsin revealed stable denaturation temperatures of 48.40 and 53.35°C, respectively. The FTIR spectra of both collagens were similar with amide regions in A, B, I, II and III. The study demonstrated that chicken feet collagen using papain isolation method is possible as commercial alternative ingredient.
Abstract: The Egyptian Bacillus thuringiensis isolate (M5) produce crystal proteins that is toxic against insects was irradiated with UV light to induce mutants. Upon testing 10 of the resulting mutants for their toxicity against cotton leafworm larvae, the three mutants 62, 64 and 85 proved to be the most toxic ones. Upon testing these mutants along with their parental isolate by SDS-PAGE analysis of spores-crystals proteins as well as vegetative cells proteins, new induced bands appeared in the three mutants by UV radiation and also they showed disappearance of some other bands as compared with the wild type isolate. Multiplex PCR technique, with five sets of specific primers, was used to detect the three types of cryI genes cryIAa, cryIAb and cryIAc. Results showed that these three genes exist, as distinctive bands, in the wild type isolate (M5) as well as in mutants 62 and 85, while the mutant 64 had two distinctive bands of cryIAb and cryIAc genes, and a faint band of cryI Aa gene. Finally, these results revealed that mutant 62 is considered as the promising mutant since it is UV resistant, highly toxic against Spodoptera littoralis and active against a wide range of Lepidopteran insects.
Abstract: The whole-cell protein-profiling technique was
evaluated for studying differences in banding pattern of three
different species of Cyanobacteria i.e. Anabaena fertilissima,
Aulosira fertilissima and Westiellopsis prolifica under the influence
of four different pesticides-2,4-D (Ethyl Ester of 2,4-Dichloro
Phenoxy Acetic Acid), Pencycuron (N-[(4-chlorophenyl)methyl]-Ncyclopentyl-
N'–phenylurea), Endosulfan (6,7,8,9,10,10hexachloro-
1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepine-3-
oxide) and Tebuconazole (1-(4-Chlorophenyl)-4,4-dimethyl-3-(1,2,4-
triazol-1-ylmethyl)pentan-3-ol). Whole-cell extracts were obtained by
sonication treatment (Sonifier cell disruptor -Branson Digital Sonifier
S-450D, USA) and were analyzed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE
analyses of the total protein profile of Anabaena fertilissima,
Aulosira fertilissima and Westiellopsis prolifica showed a linear
decrease in the protein content with increasing pesticide stress when
administered to different concentrations of 2, 4-D, Pencycuron,
Endosulfan and Tebuconazole. The results indicate that different
stressors exert specific effects on cyanobacterial protein synthesis.
Abstract: Cry j 1 is a causative substance of Japanese cedar
pollinosis, and it may deteriorate by Cry j 1 invasion to a lower
respiratory tract. We observed airborne particles containing Cry j 1 by
an immunofluorescence technique using a fluorescence microscope,
and we clarified that Cry j 1 exist as aggregates of airborne fine
particles (< 1.1 μm) in the urban atmosphere. Airborne Cry j 1 may
react with air pollutants and be denature to a substance deteriorated
Japanese cedar pollinosis. Therefore, we applied a sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate a
Cry j 1 reacted with various air pollutants by liquid phase reaction,
and calculated kinetics constants of Cry j 1 extracted from pollens
collected in various sites and airborne fine particles containing Cry j
1 by using a surface plasmon resonance (SPR) method. As a result, it
is suggested that Cry j 1 may be denatured by air pollutants during
the transportation to the urban atmosphere.
Abstract: Glutathione S-transferase was purified from human
erythrocytes and effects of some polyphenols were investigated on
the enzyme activity. The purification procedure was performed on
Glutathione-Agarose affinity chromatography after preparation of
erythrocytes hemolysate with a yield of 81%. The purified enzyme
showed a single band on the SDS-PAGE. The effects of some
poliphenolic compounds such as catechin, dopa, dopamine, progallol
and catechol were examined on the in vitro GST activity. Catechin
was determined to be inhibitor for the enzyme, but others were not
effective on the enzyme as inhibitors or activators. IC50 value -the
concentration of inhibitor which reduces enzyme activity by 50%-
was estimated to be 10 mM. Ki constants were also calculated as 6.38
± 0,70 mM with GSH substrate, and 3.86 ± 0,78 mM with CDNB
substrate using the equations of graphs for the inhibitor, and its
inhibition type was determined as non-competitive.
Abstract: Bean common mosaic necrosis virus (BCMNV) is a
potyvirus with a worldwide distribution. This virus causes serious
economic losses in Iran in many leguminoses. During 20008,
samples were collected from soybeans fields in Tehran Province.
Four isolates (S1, S2 and S3) were inoculated on 15 species of
Cucurbitaceae, Chenopodiaceae, Solanacae and Leguminosae.
Chenopodium quinoa and C. amaranticolor.
Did not developed any symptoms.all isolates caused mosaic
symptoms on Phaseolus vulgaris cv. Red Kidney and P. vulgaris cv.
Bountiful. The molecular weights of coat protein using SDS-PAGE
and western blotting were estimated at 33 kDa. Reverse transcription
polymerase chain reaction (RT-PCR) was performed using one
primer pairs designed by L. XU et al. An approximately 920 bp
fragment was amplified with a specific primer.
Abstract: Ethanol is generally used as a therapeutic reagent against Hepatocellular carcinoma (HCC or hepatoma) worldwide, as it can induce Hepatocellular carcinoma cell apoptosis at low concentration through a multifactorial process regulated by several unknown proteins. This paper provides a simple and available proteomic strategy for exploring differentially expressed proteins in the apoptotic pathway. The appropriate concentrations of ethanol required to induce HepG2 cell apoptosis were first assessed by MTT assay, Gisma and fluorescence staining. Next, the central proteins involved in the apoptosis pathway processs were determined using 2D-PAGE, SDS-PAGE, and bio-software analysis. Finally the downregulation of two proteins, AFP and survivin, were determined by immunocytochemistry and reverse transcriptase PCR (RT-PCR) technology. The simple, useful method demonstrated here provides a new approach to proteomic analysis in key bio-regulating process including proliferation, differentiation, apoptosis, immunity and metastasis.
Abstract: Proteins levels produced by bacteria may be increased
in stressful surroundings, such as in the presence of antibiotics. It
appears that many antimicrobial agents or antibiotics, when used at
low concentrations, have in common the ability to activate or repress
gene transcription, which is distinct from their inhibitory effect.
There have been comparatively few studies on the potential of
antibiotics or natural compounds in nature as a specific chemical
signal that can trigger a variety of biological functions. Therefore,
this study was focusing on the effect of essential oils from
Cymbopogon flexuosus and C. nardus in regulating proteins
production by Bacillus subtilis ATCC 21332. The Minimum
Inhibition Concentrations (MICs) of both essential oils on B. subtilis
were determined by using microdilution assay, resulting 0.2% and
1.56% for each C. flexuosus and C. nardus subsequently. The
bacteria were further exposed to each essential oils at concentration
of 0.01XMIC for 2 days. The proteins were then isolated and
analyzed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Protein profile showed that a band
with approximate size of 250 kD was appeared for the treated
bacteria with essential oils. Thus, Bacillus subtilis ATCC 21332 in
stressful condition with the presence of essential oils at low
concentration could induce the protein production.
Abstract: Lectins have a good scope in current clinical
microbiology research. In the present study evaluated the
antimicrobial activities of a D-galactose binding lectin (PnL) was
purified from the annelid, Perinereis nuntia (polychaeta) by affinity
chromatography. The molecular mass of the lectin was determined to
be 32 kDa as a single polypeptide by SDS-PAGE under both reducing
and non-reducing conditions. The hemagglutinating activity of the
PnL showed against trypsinized and glutaraldehyde-fixed human
erythrocytes was specifically inhibited by D-Gal, GalNAc,
Galβ1-4Glc and Galα1-6Glc. PnL was evaluated for in vitro
antibacterial screening studies against 11 gram-positive and
gram-negative microorganisms. From the screening results, it was
revealed that PnL exhibited significant antibacterial activity against
gram-positive bacteria. Bacillus megaterium showed the highest
growth inhibition by the lectin (250 μg/disc). However, PnL did not
inhibit the growth of gram-negative bacteria such as Vibrio cholerae
and Pseudomonas sp. PnL was also examined for in vitro antifungal
activity against six fungal phytopathogens. PnL (100 μg/mL) inhibited
the mycelial growth of Alternaria alternata (24.4%). These results
indicate that future findings of lectin applications obtained from
annelids may be of importance to life sciences.
Abstract: The present study has been taken to explore the
screening of in vitro antimicrobial activities of D-galactose-binding
sponge lectin (HOL-30). HOL-30 was purified from the marine
demosponge Halichondria okadai by affinity chromatography. The
molecular mass of the lectin was determined to be 30 kDa with a
single polypeptide by SDS-PAGE under non-reducing and reducing
conditions. HOL-30 agglutinated trypsinized and glutaraldehydefixed
rabbit and human erythrocytes with preference for type O
erythrocytes. The lectin was subjected to evaluation for inhibition of
microbial growth by the disc diffusion method against eleven human
pathogenic gram-positive and gram-negative bacteria. The lectin
exhibited strong antibacterial activity against gram-positive bacteria,
such as Bacillus megaterium and Bacillus subtilis. However, it did
not affect against gram-negative bacteria such as Salmonella typhi
and Escherichia coli. The largest zone of inhibition was recorded of
Bacillus megaterium (12 in diameter) and Bacillus subtilis (10 mm in
diameter) at a concentration of the lectin (250 μg/disc). On the other
hand, the antifungal activity of the lectin was investigated against six
phytopathogenic fungi based on food poisoning technique. The lectin
has shown maximum inhibition (22.83%) of mycelial growth of
Botrydiplodia theobromae at a concentration of 100 μg/mL media.
These findings indicate that the lectin may be of importance to
clinical microbiology and have therapeutic applications.