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: In recent years, there has been an increasing interest
toward the use of bovine genotyped embryos for commercial embryo
transfer programs. Biopsy of a few cells in morulla stage is essential
for preimplantation genetic diagnosis (PGD). Low amount of DNA
have limited performing the several molecular analyses within PGD
analyses. Whole genome amplification (WGA) promises to eliminate
this problem. We evaluated the possibility and performance of an
improved primer extension preamplification (I-PEP) method with a
range of starting bovine genomic DNA from 1-8 cells into the WGA
reaction. We optimized a short and simple I-PEP (ssI-PEP) procedure
(~3h). This optimized WGA method was assessed by 6 loci specific
polymerase chain reactions (PCRs), included restriction fragments
length polymorphism (RFLP). Optimized WGA procedure possesses
enough sensitivity for molecular genetic analyses through the few
input cells. This is a new era for generating characterized bovine
embryos in preimplantation stage.
Abstract: Y chromosome microdeletions are the most common
genetic cause of male infertility and screening for these
microdeletions in azoospermic or severely oligospermic men is now
standard practice. Analysis of the Y chromosome in men with
azoospermia or severe oligozoospermia has resulted in the
identification of three regions in the euchromatic part of the long arm
of the human Y chromosome (Yq11) that are frequently deleted in
men with otherwise unexplained spermatogenic failure. PCR analysis
of microdeletions in the AZFa, AZFb and AZFc regions of the
human Y chromosome is an important screening tool. The aim of this
study was to analyse the type of microdeletions in men with fertility
disorders in Slovakia. We evaluated 227 patients with azoospermia
and with normal karyotype. All patient samples were analyzed
cytogenetically. For PCR amplification of sequence-tagged sites
(STS) of the AZFa, AZFb and AZFc regions of the Y chromosome
was used Devyser AZF set. Fluorescently labeled primers for all
markers in one multiplex PCR reaction were used and for automated
visualization and identification of the STS markers we used genetic
analyzer ABi 3500xl (Life Technologies). We reported 13 cases of
deletions in the AZF region 5,73%. Particular types of deletions were
recorded in each region AZFa,b,c .The presence of microdeletions in
the AZFc region was the most frequent. The study confirmed that
percentage of microdeletions in the AZF region is low in Slovak
azoospermic patients, but important from a prognostic view.
Abstract: Bone marrow-derived stem cells have been widely
studied as an alternative source of stem cells. Mesenchymal stem
cells (MSCs) were mostly investigated and studies showed MSCs can
promote neurogenesis. Little is known about the non-mesenchymal
mononuclear cell fraction, which contains both hematopoietic and
nonhematopoietic cells, including monocytes and endothelial
progenitor cells. This study focused on unfractionated bone marrow
mononuclear cells (BMMCs), which remained 72 h after MSCs were
adhered to the culture plates. We showed that BMMC-conditioned
medium promoted morphological changes of human SH-SY5Y
neuroblastoma cells from an epithelial-like phenotype towards a
neuron-like phenotype as indicated by an increase in neurite
outgrowth, like those observed in retinoic acid (RA)-treated cells.
The result could be explained by the effects of trophic factors
released from BMMCs, as shown in the RT-PCR results that
BMMCs expressed nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF).
Similar results on the cell proliferation rate were also observed
between RA-treated cells and cells cultured in BMMC-conditioned
medium, suggesting that cells creased proliferating and differentiated
into a neuronal phenotype. Using real-time RT-PCR, a significantly
increased expression of tyrosine hydroxylase (TH) mRNA in SHSY5Y
cells indicated that BMMC-conditioned medium induced
catecholaminergic identities in differentiated SH-SY5Y cells.
Abstract: Before performing polymerase chain reactions (PCR), a feasible primer set is required. Many primer design methods have been proposed for design a feasible primer set. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this paper, a particle swarm optimization (PSO) algorithm is proposed to solve primer design problems associated with providing a specific product for PCR experiments. A test set of the gene CYP1A1, associated with a heightened lung cancer risk was analyzed and the comparison of accuracy and running time with the genetic algorithm (GA) and memetic algorithm (MA) was performed. A comparison of results indicated that the proposed PSO method for primer design finds optimal or near-optimal primer sets and effective PCR products in a relatively short time.
Abstract: The cDNA encoding the 326 amino acids of a Class I
basic chitinase gene from Leucaena leucocephala de Wit (KB3,
Genbank accession: AAM49597) was cloned under the control of
CaMV35S promoter in pCAMBIA 1300 and transferred to
Koshihikari. Calli of Koshihikari rice was transformed with
agrobacterium with this construct expressing the chitinase and β-
glucouronidase (GUS). The frequencies of calli 90 % has been
obtained from rice seedlings cultured on NB medium. The high
regeneration frequencies, 74% was obtained from calli cultured on
regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at
25°C. Various factors were studied in order to establish a procedure
for the transformation of Koshihikari Agrobacterium tumefaciens.
Supplementation of 50 mM acetosyringone to the medium during
coculivation was important to enhance the frequency to transient
transformation. The 4 week-old scutellum-derived calli were
excellent starting materials. Selection medium based on NB medium
supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were
an optimized medium for selection of transformed rice calli. The
percentage of transformation 70 was obtained. Recombinant calli and
regenerated rice plants were checked the expression of chitinase and
gus by PCR, northern blot gel, southern blot gel, and gus assay.
Chitinase and gus were expressed in all parts of recombinant rice.
The rice line expressing the KB3 chiitnase was more resistant to the
blast fungus Fusarium monoliforme than control line.
Abstract: research goal was to determine the expression levels cDNA of brain embrio at gestation days 10 (GD-10). The Electroforesis DNA results showed that GAPDH, Fibronectin1, Ncam1, Tenascin, Vimentin, Neurofilament heavy, Neurofilament medium and Neurofilament low were 447 bp, 462 bp, 293 bp. 416 bp, 327 bp, 301 bp, 398 bp and 289 bp. Result of real-time RT-PCR on brain Embryo at gestation days 10 showed that the expression of copy gen Fibronectin 36 copies, Ncam 21,708 copies; Tenascin 24,505 copies; Vimentin 538,554 copies; Neurofilament heavy 2,419 copies; Neurofilament medium 92,928 copies; Neurofilament low 125,809 copies. Vimentin expressed gene copies is very high compared with other gene copies. This condition are caused by Vimentin, that contribute to proliferate of brain development. The vimentin role to cell proliferation of brain.
Abstract: In this paper, we propose the pre-processor based on
the Evidence Supporting Measure of Similarity (ESMS) filter and also
propose the unified fusion approach (UFA) based on the general
fusion machine coupled with ESMS filter, which improve the
correctness and precision of information fusion in any fields of
application. Here we mainly apply the new approach to Simultaneous
Localization And Mapping (SLAM) of Pioneer II mobile robots. A
simulation experiment was performed, where an autonomous virtual
mobile robot with sonar sensors evolves in a virtual world map with
obstacles. By comparing the result of building map according to the
general fusion machine (here DSmT-based fusing machine and
PCR5-based conflict redistributor considereded) coupling with ESMS
filter and without ESMS filter, it shows the benefit of the selection of
the sources as a prerequisite for improvement of the information
fusion, and also testifies the superiority of the UFA in dealing with
SLAM.
Abstract: The full length mitochondrial small subunit ribosomal
(mt-rns) gene has been characterized for Ophiostoma novo-ulmi
subspecies americana. The gene was also characterized for
Ophiostoma ulmi and a group II intron was noted in the mt-rns gene
of O. ulmi. The insertion in the mt-rns gene is at position S952 and it
is a group IIB1 intron that encodes a double motif LAGLIDADG
homing endonuclease from an open reading frame located within a
loop of domain III. Secondary structure models for the mt-rns RNA
of O. novo-ulmi subsp. americana and O. ulmi were generated to
place the intron within the context of the ribosomal RNA. The in vivo
splicing of the O.ul-mS952 group II intron was confirmed with
reverse transcription-PCR. A survey of 182 strains of Dutch Elm
Diseases causing agents showed that the mS952 intron was absent in
what is considered to be the more aggressive species O. novo-ulmi
but present in strains of the less aggressive O. ulmi. This observation
suggests that the O.ul-mS952 intron can be used as a PCR-based
molecular marker to discriminate between O. ulmi and O. novo-ulmi
subsp. americana.
Abstract: A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.
Abstract: D-erythro-cyclohexylserine (D
chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion
system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene
was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on
genomic sequence of Shinorhizobium meliloti
Sequence analysis of the cloned DNA fragment revealed one
open-reading frame of 1059 bp and 386 amino acids. This putative
D-TA gene was cloned into NdeI and EcoRI (pEnsi
His-tag sequence or BamHI (pEnsi-DTA[2])
sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA[1] compared to E. coli BL21(DE3) transformed with
pEnsi-DTA[2]. When the cells expressing the wild
used for D-TA enzyme activity, 12 mM glycine was successfully
detected in HPLC analysis. Moreover, the whole cells harbouring the
recombinant D-TA was able to synthesize D-erythro
of 0.6 mg/ml in a batch reaction.
Abstract: Type 2 diabetes mellitus (T2DM) is a complex
metabolic disorder that characterized by the presence of high glucose
in blood that cause from insulin resistance and insufficiency due to
deterioration β-cell Langerhans functions. T2DM is commonly
caused by the combination of inherited genetic variations as well as
our own lifestyle. Metallothionein (MT) is a known cysteine-rich
protein responsible in helping zinc homeostasis which is important in
insulin signaling and secretion as well as protection our body from
reactive oxygen species (ROS). MT scavenged ROS and free
radicals in our body happen to be one of the reasons of T2DM and its
complications. The objective of this study was to investigate the
association of MT1A and MT2A polymorphisms between T2DM and
control subjects among Malay populations. This study involved 150
T2DM and 120 Healthy individuals of Malay ethnic with mixed
genders. The genomic DNA was extracted from buccal cells and
amplified for MT1A and MT2A loci; the 347bp and 238bp banding
patterns were respectively produced by mean of the Polymerase
Chain Reaction (PCR). The PCR products were digested with Mlucl
and Tsp451 restriction enzymes respectively and producing
fragments lengths of (158/189/347bp) and (103/135/238bp)
respectively. The ANOVA test was conducted and it shown that there
was a significant difference between diabetic and control subjects for
age, BMI, WHR, SBP, FPG, HBA1C, LDL, TG, TC and family
history with (P0.05). The genotype
frequency for AA, AG and GG of MT1A polymorphisms was 72.7%,
22.7% and 4.7% in cases and 15%, 55% and 30% in control
respectively. As for MT2A, genotype frequency of GG, GC and CC
was 42.7%, 27.3% and 30% in case and 5%, 40% and 55% for
control respectively. Both polymorphisms show significant difference
between two investigated groups with (P=0.000). The Post hoc test
was conducted and shows a significant difference between the
genotypes within each polymorphism (P=0. 000). The MT1A and
MT2A polymorphisms were believed to be the reliable molecular
markers to distinguish the T2DM subjects from healthy individuals in
Malay populations.
Abstract: It is believed that DNA damaging toxic metabolites contributes to the development of different pathological conditions. To prevent harmful influence of toxic agents, cells developed number of protecting mechanisms, such as enzymatic reaction of detoxification of reactive metabolites and repair of DNA damage. The aim of the study was to examine the association between polymorphism of GSTT1/GSTM1 and XRCC1/3 genes and coronary artery disease (CAD) incidence. To examine a polymorphism of these genes in CAD susceptibility in patients and controls, PCR based genotyping assay was performed. For GST genes, frequency of GSTM1 null genotype among CAD affected group was significantly increased than in control group (P0.1). We found that neither XRCC1 Arg399Gln nor XRCC3 Thr241Met were associated with CAD risk. Obtained data suggests that GSTM1 null genotype carriers are more susceptible to CAD development.
Abstract: As part of national epidemiological survey on bovine
viral diarrhea virus (BVDV), a total of 274 dejecta samples were
collected from 14 cattle farms in 8 areas of Xinjiang Uygur
Autonomous Region in northwestern China. Total RNA was extracted
from each sample, and 5--untranslated region (UTR) of BVDV
genome was amplified by using two-step reverse
transcriptase-polymerase chain reaction (RT-PCR). The PCR products
were subsequently sequenced to study the genetic variations of BVDV
in these areas. Among the 274 samples, 33 samples were found
virus-positive. According to sequence analysis of the PCR products,
the 33 samples could be arranged into 16 groups. All the sequences,
however, were highly conserved with BVDV Osloss strains. The virus
possessed theses sequences belonged to BVDV-1b subtype by
phylogenetic analysis. Based on these data, we established a typing
tree for BVDV in these areas. Our results suggested that BVDV-1b
was a predominant subgenotype in northwestern China and no
correlation between the genetic and geographical distances could be
observed above the farm level.
Abstract: The p53 tumor suppressor gene plays two important
roles in genomic stability: blocking cell proliferation after DNA
damage until it has been repaired, and starting apoptosis if the
damage is too critical. Codon 72 exon4 polymorphism (Arg72Pro) of
the P53 gene has been implicated in cancer risk. Various studies have
been done to investigate the status of p53 at codon 72 for arginine
(Arg) and proline (Pro) alleles in different populations and also the
association of this codon 72 polymorphism with various tumors. Our
objective was to investigate the possible association between P53
Arg72Pro polymorphism and susceptibility to colorectal cancer
among Isfahan and Chaharmahal Va Bakhtiari (a part of south west
of Iran) population. We investigated the status of p53 at codon 72 for
Arg/Arg, Arg/Pro and Pro/Pro allele polymorphisms in blood
samples from 145 colorectal cancer patients and 140 controls by
Nested-PCR of p53 exon 4 and digestion with BstUI restriction
enzyme and the DNA fragments were then resolved by
electrophoresis in 2% agarose gel. The Pro allele was 279 bp, while
the Arg allele was restricted into two fragments of 160 and 119 bp.
Among the 145 colorectal cancer cases 49 cases (33.79%) were
homozygous for the Arg72 allele (Arg/Arg), 18 cases (12.41%) were
homozygous for the Pro72 allele (Pro/Pro) and 78 cases (53.8%)
found in heterozygous (Arg/Pro).
In conclusion, it can be said that p53Arg/Arg genotype may be
correlated with possible increased risk of this kind of cancers in south
west of Iran.
Abstract: The objective was to determine the single gene and
interaction effect of composite genotype of beta-kappa casein and
DGAT1 gene on milk yield (MY) and milk composition, content of
milk fat (%FAT), milk protein (%PRO), solid not fat (%SNF), and
total solid (%TS) in crossbred Holstein cows. Two hundred and
thirty- one cows were genotyped with PCR-RFLP for DGAT1 and
composite genotype data of beta-kappa casein from previous work
were used. Two model, (1), and (2), was used to estimate single gene
effect, and interaction effect on the traits, respectively. The
significance of interaction effects on all traits were detected. Most
traits have consistent pattern of significant when model (1), and (2)
were compared, except the effect of composite genotype of betakappa
casein on %FAT, and the effect of DGAT1 on MY, which the
significant difference was detected in only model (1).The results
suggested that when the optimum of all traits was necessary,
interaction effect should be concerned.
Abstract: Attachment of the circulating monocytes to the
endothelium is the earliest detectable events during formation of
atherosclerosis. The adhesion molecules, chemokines and matrix
proteases genes were identified to be expressed in atherogenesis.
Expressions of these genes may influence structural integrity of the
luminal endothelium. The aim of this study is to relate changes in the
ultrastructural morphology of the aortic luminal surface and gene
expressions of the endothelial surface, chemokine and MMP-12 in
normal and hypercholesterolemic rabbits. Luminal endothelial
surface from rabbit aortic tissue was examined by scanning electron
microscopy (SEM) using low vacuum mode to ascertain
ultrastructural changes in development of atherosclerotic lesion. Gene
expression of adhesion molecules, MCP-1 and MMP-12 were studied
by Real-time PCR. Ultrastructural observations of the aortic luminal
surface exhibited changes from normal regular smooth intact
endothelium to irregular luminal surface including marked globular
appearance and ruptures of the membrane layer. Real-time PCR
demonstrated differentially expressed of studied genes in
atherosclerotic tissues. The appearance of ultrastructural changes in
aortic tissue of hypercholesterolemic rabbits is suggested to have
relation with underlying changes of endothelial surface molecules,
chemokine and MMP-12 gene expressions.
Abstract: Brucellosis is a zoonotic disease; its symptoms and appearances are not exclusive in human and its traditional diagnosis is based on culture, serological methods and conventional PCR. For more sensitive, specific detection and differentiation of Brucella spp., the real time PCR method is recommended. This research has performed to determine the presence and prevalence of Brucella spp. and differentiation of Brucella abortus and Brucella melitensis in house mouse (Mus musculus) in west of Iran. A TaqMan analysis and single-step PCR was carried out in total 326 DNA of Mouse's spleen samples. From the total number of 326 samples, 128 (39.27%) gave positive results for Brucella spp. by conventional PCR, also 65 and 32 out of the 128 specimens were positive for B. melitensis, B. abortus, respectively. These results indicate a high presence of this pathogen in this area and that real time PCR is considerably faster than current standard methods for identification and differentiation of Brucella species. To our knowledge, this study is the first prevalence report of direct identification and differentiation of B. abortus and B. melitensis by real time PCR in mouse tissue samples in Iran.
Abstract: A lot of recent research have spoken on the relation
between the increase of the homocysteinemia and some kinds of
cancer . For that, our study was based on the research of a possible
relation between the increase of the concentration of this amino-acid
in the plasma and the appearance of the disease of the Acute
Lymphoblastic Leukaemia in a part of Algerian children with Berber
origin in the East of Algeria . The study has done on 47 ill persons
with an average age of (09±06 ) years , with whom the disease has
diagnosed by blood and marrow examination in the hospital of blood
diseases in the CHU of Batna, and on 194 healthy witnesses of the
same age. The two groups were benefited by a dosage of the
concentration of the homocysteine vitamin B9 ,vitamin B12 , and
also of the study of special polymorphisms of indispensable enzymes
in the metabolism of this acid , and that by the use of the method (
Light cycler ) Real time PCR , on the following enzymes : MS (
C2756G ), MSR ( A66G ) ,MTHFR1 ( C677T ) and MTHFR2
(A1298C). The obtained results have revealed that the rate of the
homozygote muted genotype is the less frequent in the two groups ,
and that exist at list one genotype of each enzyme in the ill group and
in which the percentage exceed with remarkable way the same
genotype in the healthy group and we notice specially the muted
genotype GG of -the methionine synthetase-and the form TT of the
enzyme – methyline tetra hydrofolate reductase – We notice the
existence of considerable number of genotypes in the ill group lied
with characteristic increase of this Amino-acid ,and that for the
reduction of the biologic activity of these enzymes which become
inefficient in the transfer of the homocysteine into the methionine
and cause the diminution of the biologic activity of these enzymes
and with consequence the reduction of the percentage of methylic
radicals in the DNA of studied genes and that lead to the increase of
the activity and the capacity of transcription , and it-s so probably
that this last one is one of the factors of this disease especially if we
know that the specific check-up of vitamins is normal and similar in
the two groups , which ovoid the hypothesis of the reduction of
vitamins . We notice also that the heterozygote genotype is the less in
the sick category except the MTHFR2. Wild genotype is more
frequent in the witness group except MSR. Even these results are
partials; they open a new way in the genetic diagnosis of this
malicious disease which allow a precocious diagnosis and the use of
an effective and appropriated treatment in the same time.
Abstract: MMR vaccine failure had been reported globally and
here we report that it occurs now in India. Samples were collected from clinically suspected mumps cases were subjected for anti
mumps antibodies, virus isolation, RT-PCR, sequencing and
phylogenetic tree analysis. 56 samples collected from men and women belonging to various age groups. 30 had been vaccinated and
the status of 26 patients was unknown. 28 out of 30 samples were
found to be symptomatic and positive for Mumps IgM, indicating
active mumps infection in 93.4% of the vaccinated population. A
phylogenetic tree comparison of the clinical isolate is shown to be genotype C which is distinct from vaccine strain. Our study clearly sending warning signs that MMR vaccine is a failure and it needs to be revamped for the human use by increasing its efficacy and efficiency.