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Volume 10 - 2006


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1-12

Low-usage codons and rare codons of Escherichia coli

Author(s): Dr. Dequan Chen,

Abstract: In Escherichia coli (E. coli), a low-usage codon is defined as a codon that is used rarely or infrequently in the genome with usage frequency lower than the smallest value (or frequency cut-off) among the usage frequencies of non-degenerate codons (Met codon AUG and Trp codon UGG) and the optimal codons for amino acids Leu, Ile, Val, Ser, Pro, Thr, Ala, Arg, Gly and Gln that have 2 or more degenerate codons with each having specific corresponding cognate tRNA for the optimal codon. A rare codon (RC), an infrequent codon or a minor codon is equivalently defined as a synonymous codon or a stop codon that is not only used rarely or infrequently in a genome but also decoded by a low-abundant tRNA (rare tRNA) or other factor(s) in an organism. The translational rate for a sense RC is much lower than that for a common (major) codon due to tRNA availability. A low-usage codon is not necessarily a RC, e.g., Cys codons UGU and UGC, Thr codons ACU and ACG, or His codons CAC and CAU are not rare codons of E. coli. However, a RC is definitely a low-usage codon. In E. coli, there are about 30 low-usage synonymous sense codons but only 20 of them are determined to be the bacterial RCs including 7 (AGG, AGA, CGA, CUA, AUA, CCC and CGG) used at a frequency of < 0.5% (Group I) and 13 (ACA, CCU, UCA, GGA, AGU, UCG, CCA, UCC, GGG, CUC, CUU, UCU and UUA) used at a frequency of > 0.5% (Group II). Studies have demonstrated that all the RCs in Group I and the first 6 RCs in Group 2 can cause translational problems in E. coli.

Keywords: low-usage codon, rare codon, common codon, major codon, codon usage, infrequent codon, rare tRNA, codon optimization, and rare tRNA supplementation



13-16

Structural analysis of the elongated part of an abnormal hemoglobin “Hemoglobin Cranston”

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Haemoglobin variants in which a frameshift results in chain elongation are unusual. Haemoglobin Cranston (HbCranston) is an unstable haemologin firstly with abnormal elongation. Concerning the pathogenesis of HbCranston, the insertion of the repeated pair nucleotide pair AG into β mRNA between the triplet codon of 144 Lysine (AAG) and 145 Tyrosine (UAU) is the main abnormality. It is assumed to be due to an insertion of the dinucleotide CA into codon 146 [CACCA(CA)C] which abolishes the normal stop codon at position 147 (Bunn et al, 1975). This abnormality causes a frameshift, which results in elongation of the β chain amino acids. Here, the author performs a bioinformatic analysis to study the secondary and tertiary structures of those elongated amino acid sequences. Answering this question, a computer-based study for protein structure modeling is performed. According to this study, the secondary structure analysis of the elongated part of Hb Cranston showed eleven additional helices to the normal β globin chains. Based on this information, the main alteration in the Hb Cranston might be due to the additional helices in the elongated part. Concerning the tertiary structure, the increase of folds, accompanied with the aberration in secondary structure of globin in Hb Cranston can be identified.

Keywords: Hb Cranston, structure



17-30

Delivery of human apolipoprotein (apo) E to liver by an [E1–, E3–, polymerase–, pTP–] adenovirus vector containing a liver-specific promoter inhibits atherogenesis in immunocompetent apoE-deficient mice

Author(s): Dr. George Dickson,

Abstract: Recombinant adenovirus (rAd)-mediated apoE gene transfer to the liver of apoE-/- mice is anti-atherogenic. However, first generation rAd vectors were associated with immune clearance of transduced hepatocytes, while an improved [E1-, E3- polymerase-] adenovirus vector that persisted in the liver, had transient effects due to cellular shutdown of the cytomegalovirus (CMV) promoter (Ad-CMV-apoE). Here, we utilise an improved class of rAd vector with multiple deletions in the E1, E3, polymerase and pTP (pre-terminal protein) genes, which contains a modular synthetic liver-specific promoter (LSP) to drive expression of the human apoE cDNA (Ad-LSP-apoE) for hepatic gene transfer. Approximately 1 year old apoE-/- mice were injected intravenously with 4x1010 virus particles of either Ad-LSP-apoE or Ad-CMV-apoE. Animals were monitored for plasma apoE, total plasma cholesterol and plasma lipoprotein distribution. The effect of Ad-LSP-apoE on atheroma progression was assessed in animals killed at 8 and 28 weeks after the injections. Ad-LSP-apoE vector administration gave sustained, though low, levels of plasma apoE throughout the study period without inducing a humoral immune response, but failed to reduce plasma cholesterol or normalize the adverse lipoprotein profile. Animals killed 8 weeks after the injections, demonstrated no significant retardation of atherosclerosis, whereas aortic lesions in those killed at 28 weeks were significantly reduced by 30% (P<0.006) compared to untreated animals. In summary, the combination of a multiply deleted rAd vector with a liver-specific promoter provided sustained low levels of plasma apoE, resulting in significant retardation of aortic atherosclerotic lesions.

Keywords: Atherosclerosis; Apolipoprotein E; Gene Therapy; Adenovirus



31-40

Naturally occurring translational models for development of cancer gene therapy

Author(s): Dr. Jaime F. Modiano,

Abstract: Most cancer deaths occur from metastatic spread of cancer cells. Immunotherapy and gene therapy are appealing modalities to treat cancer, not only because tumors that are resistant to conventional treatment such as radiation and chemotherapy can be treated using immunologic and genetic approaches, but also because these modalities can reach distant metastases and tumors that are inaccessible for conventional treatment. One gene therapy-based immunologic approach that has shown preclinical promise in laboratory animals is the use of Fas ligand (FasL) gene transfer. FasL promotes tumor cell killing directly and indirectly, and it induces reliable antitumor immune responses that protect animals against subsequent tumor challenge. Yet, despite the unquestioned benefits to study mechanistic questions, factors such as size, pharmacokinetic distribution, and route of administration preclude precise extrapolation of safety data from laboratory mice to humans. We have used spontaneous cancers of dogs as intermediaries for translational studies because the size and physiology of dogs, as well as the natural history of homologous tumors in this species resemble those of humans more closely than rodent models created in the laboratory. Here, we use appendicular osteosarcoma (OS) as an example to document clinical and biological similarities between the disease in dogs and humans. Specifically, we underscore the unique properties of this model to develop therapy approaches prior to translation into clinical trials of human cancer patients.

Keywords: Gene Therapy, Immunotherapy, Fas Ligand, Osteosarcoma, Canine



41-54

CDK inhibitors in 3D: Problems with the drugs, their development plans or their linkage to disease?

Author(s): Dr. Andrew Hughes,

Abstract: Targeting the cell cycle is an attractive anticancer strategy, as its dysregulation is a common, if not ubiquitous, occurrence in tumour development. Cell-cycle control is achieved by the interaction of a complex set of enzymes and other proteins, including the family of protein kinases known as cyclin-dependent kinases (CDKs) and the protein product of the tumour suppressor gene Retinoblastoma. CDKs are required for the orderly progression of cells through the cell cycle and hyperactivation of the CDKs in tumour development, as well as mutation and overexpression of these proteins, is common. Several compounds collectively known as ‘CDK inhibitors’ have been in clinical trials for a number of years. Although often grouped together, they differ in their molecular and cellular modes of action. Several agents in this group, such as flavopiridol, E7070 and UCN-01, have multiple CDK and non- CDK targets or inhibit upstream regulators of the CDKs, whereas other CDK inhibitors, such as CYC-202, appear to target CDK1 and CDK2 more specifically. Most CDK inhibitors are administered intravenously and various schedules have been explored in the clinic. Although generally well tolerated, CDK inhibitors as monotherapy have given mixed efficacy results, possibly due to problems related to specificity or in dosing/scheduling leading to suboptimal exposure. A lack of pharmacodynamic end points, together with the multiple cellular targets of some agents, has made the assessment of ‘CDK inhibition’ and its contribution to antitumour effect in the clinic difficult. However, recent pharmacokinetic studies are examining dosing and scheduling regimens, and novel markers of drug activity and patient suitability are being developed. In addition, newer, specifically targeted oral agents may prove more effective, less toxic and more amenable to optimisation in clinical trials.

Keywords: CDK, CDK inhibitor, cell cycle, pRb, flavopiridol, UCN-01, CYC202, E7070, BMS 387032



55-60

Antioxidative gene therapy using superoxide dismutase in ischemia-reperfusion injury of testes in rats

Author(s): Dr. Peter Celec,

Abstract: Ischemia-reperfusion injury (IRI) is associated with increased production of reactive oxygen species and thus with oxidative stress. Antioxidative pre-treatment prevents the free radical induced tissue impairment in experiment, as has been shown previously. However, clinical use requires long-term treatment with high doses of antioxidants. Gene therapy using safe vector constructs provides a useful tool for gene transfer in vivo. To evaluate the effects of superoxide dismutase (SOD) gene therapy on oxidative stress induced tissue impairment in an experimental model of IRI of testes. Male Wistar rats (n=18) were pre-treated either by single intratesticular injection of 500 μg of plasmid pcDNA3 containing the Mn-SOD cDNA or by adequate volume of saline two days prior to the IRI. Ischemia (30 minutes) was induced by torsion of a random testis in each rat. After 30 minutes of reperfusion (detorsion), both testes were removed. Malondialdehyde (MDA) as major product of lipoperoxidation were measured in testes homogenates. Samples were also analysed by electron microscopy. Although not reaching the level of statistical significance our results show that IRI increased the oxidative stress induced tissue impairment and SOD gene pre-treatment could partly compensate and reduce the MDA level. This decrease is even superior without IRI. This study does not provide any evidence, but indicates the possibilities of antioxidative gene therapy in IRI of testes. Further larger studies should prove the efficiency of this approach on other organs using wider palette of markers of tissue impairment.

Keywords: gene therapy, superoxide dismutase, ischemia-reperfusion injury, testes, torsion, thiobarbituric acid reacting substances



61-70

Viral vectors in pancreatic cancer gene therapy

Author(s): Dr. Min Li,

Abstract: Pancreatic cancer is the fourth leading cause of cancer related deaths, and effective diagnostic and therapeutic strategies are lacking. Molecular research seeking genetic events and signaling pathways that are crucial in pancreatic carcinogenesis holds promise for the development of effective gene therapy strategies. The success of such strategies will depend on efficient, specific, and safe gene delivery to target cells as well as improved target cell- specific gene expression. Adenoviral vectors and retro/lenti-viral vectors have gained significant popularity in cancer gene therapy strategies because of their superior gene transfer efficiency and stable gene expression property, respectively. Many studies have been done to deliver tumor suppressor genes or suicide genes using adenovirus or retro/lenti-virus vectors to treat pancreatic cancer. Another promising therapeutic strategy for pancreatic cancer is the use of conditionally replicating (oncolytic) viruses. These viruses can selectively replicate in cancer cells, and their progeny viruses subsequently spread to surrounding cells, therefore achieving a large scale of viral infection. Several viruses, including adenovirus, herpes simplex virus (HSV), and reovirus have been modified for oncolytic purpose, and incorporation of extra tumoricidal strategies such as enzyme-directed pro-drug and fusogenic viral glycoproteins can further potentiate their anti-tumor capacity. This review provides a brief overview of gene therapy strategies using different viral vectors and anti-tumor activities of oncolytic viruses for pancreatic cancer treatment.

Keywords: Viral vectors, Gene therapy, Pancreatic cancer, Cancer phenotype, Immunotherapy, Suicide gene therapy, Antiangiogenic therapy, Transductional targeting, Transcriptional targeting, Retrovirus, Adenovirus, Toxicity, Oncolytic viral therapy, Reovirus,



71-94

Design of functional dendritic polymers for application as drug and gene delivery systems

Author(s): Dr. Zili Sideratou,

Abstract: The present review deals with the design and preparation of functional and multifunctional dendrimeric and hyperbranched polymers (dendritic polymers), in order to be employed as drug and gene delivery systems. In particular, using as starting materials known and well-characterized basic dendritic polymers, the review discusses the kind of structural modifications that these polymers were subjected for preparing nanocarriers of low toxicity, high encapsulating capacity, specificity to certain biological cells and transport ability through their membranes. Due to the great number of external groups of dendritic polymers either functionalization or multifunctionalization can occur, providing products that fulfill one or more of the requirements that an effective drug carrier should exhibit. A common feature of these dendritic polymers is the exhibition of the so-called polyvalent interactions, while for the multifunctional derivatives a number of targeting ligands determines specificity, other groups secure stability in biological milieu, while others facilitate their transport through cell membranes. In addition, for gene delivery applications these multifunctional systems should be or become cationic in the biological environment for the formation of complexes with the negatively charged genetic material.

Keywords: Dendrimers, Hyperbranched Polymers, Dendritic Polymers, Nanocarriers, Drug Delivery System, Gene Delivery



95-100

The role of BRCA1 AND BRCA2 in hereditary breast cancer

Author(s): Dr. Athina Christopoulou,

Abstract: BRCA1 and BRCA2 account for most cases of hereditary breast cancer in the United States and Europe. These are suppressor genes that are inherited in an autosomal dominant fashion. Several studies showed that the histologic and molecular phenotype of BRCA-associated tumors is different from that of nonhereditary tumors. There is a difference in steroid receptor status between BRCA1 and 2 tumors regard to chemoprevention of breast cancer with antiestrogenes. 93-100% of BRCA2 associated breast cancers are ER/PR+. Breast cancers associated with BRCA1 mutations are frequently of a higher grade and are hormone receptor-negative in one third of them. A higher proportion of cancers related to a BRCA1 mutation have atypical or typical medullary histologic features. The lifetime cumulative risk of invasive breast cancer for individuals with BRCA1 or BRCA2 mutations ranges from 50% to 87%. Familial breast cancer, however, accounts for fewer than 10% of all breast cancers, and BRCA1-related and BRCA2-related familial disease constitutes only two-thirds to three-fourths of these cases. Among women younger than 35 years old with breast cancer, 10% to 15% have a BRCA1 mutation. Woman with BRCA 1/2 mutations already affected by the disease have a risk, to age 70, of contralateral breast cancer that ranges between 50% and 64%. It has been difficult to determine whether germline BRCA 1/2 status has an effect on breast cancer outcome and the results from several studies remain controversial. There are preliminary data that BRCA 1/2 related tumors may have a faster growth rate than sporadic tumors. In these women prophylactic mastectomy, chemoprevention with tamoxifen or prophylactic oophorectony are reasonable options. Genetic testing for BRCA 1/2 mutations should be done in those with a significant family history of breast or ovarian cancer, those with a diagnosis of breast or ovarian cancer below 50 years of age and those with a blood relative who is known to have a mutation in BRCA 1 or 2. Ongoing clinical trials will determine who the optimal subjects are for screening, how screening and counseling should be conducted and what type of societal involvement is needed so that genetic screening can be used without exposing the subject to unexpected risks and consequences.

Keywords: hereditary breast cancer, BRCA1, BRCA2



101-108

The analysis of dose response curve comes in useful for the assembly of multi-siRNAs expressing cassettes

Author(s): Dr. Giuseppe Rainaldi,

Abstract: The construction of vectors that would allow the simultaneous expression of multiple siRNAs targeted against different genes is hampered by the competition between siRNAs. In this work, the simultaneous knock-down of four genes involved in smooth muscle cells activation, migration and proliferation was considered. We used the knock- down of EGFP reporter assay to evaluate the dose response curves of the four shRNA expressing plasmids. We found that each siRNA reached the highest acitivity (as evaluated from the plateau phase) with different kinetics (as evaluated from the KD). Due the specificity of KD, the mono-specific plasmids were tested against their targets by the addiction of saturating amounts of each of the other shRNA-expressing plasmid. In this way, stronger from weaker shRNAs were distinguished and KD seemed to account for it. Moreover, when stronger shRNAs were assembled, the resulting plasmid was able to simultaneously transcribe active shRNAs genes. These results indicate that expression cassettes for different siRNAs having similar KD can be efficiently and rapidly assembled into multi-specific multi-siRNA plasmids. A practical correlate of these observations is that, in order to obtain effective multi-gene knock down, only siRNAs with similar inhibitory kinetics need to be delivered to the cells.

Keywords: simultaneous gene expression knock-down, competition among siRNAs, multi-shRNA vectors



109-112

The isolation of chlamydia pneumoniae in atherosclerosis patients in Iran by PCR method

Author(s): Dr. Fatemeh Fallah,

Abstract: Cardiovascular disease (CAD) is the leading cause of death in developed countries. The cause is multifactorial. A substantial proportion of patients with CAD do not have traditional risk factors. Infectious diseases may play a role in these cases, or they may intensify the effect of the risk factors. The association of CAD and Chlamydia pneumoniae infection is firmly established, but causality is yet to be proven. We investigated their presence in carotid atherosclerotic plaques. 102 plaque atherosclerotic in dead patients were available for examination in Tehran, Iran. The highly sensitive polymerase chain reaction method was employed with primers specific for this agent. The presence of Chlamydia DNA was detected in 23 (22%) out of 102 examined samples. The presence of Chlamydia DNA in these patients supports the hypothesis that this agent has an association with atherosclerosis.

Keywords: ChlamydiaPneumoniae,atherosclerosis,PCRamplification, Cardiovasculardisease



113-122

Using N-(2-hydroxypropyl) methacrylamide copolymer drug bioconjugate as a novel approach to deliver a Bcl-2-targeting compound HA14-1 in vivo

Author(s): Dr. Ray M. Lee,

Abstract: Bcl-2 plays a critical role in regulation of apoptosis and tumor pathogenesis; thus it’s a good therapeutic target for cancer. Small compounds blocking Bcl-2 have been identified but their efficacy in vivo has hardly been demonstrated. We developed water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing a Bcl-2 targeting compound HA14-1. Their efficacy was confirmed in cell lines, and tested in a tumor xenograft model. Intraperitoneal injected copolymer HA14-1 bioconjugates suppressed tumor growth by 50%. Using FITC as a marker to trace biodistribution, we demonstrated that the concentration of the copolymer was sufficient to induce apoptosis. This was confirmed by the presence of activated caspase 9 in tumor treated with the copolymer HA14-1 bioconjugate, but not in normal organs or tumor treated with a control polymer. No toxicity was observed in liver and kidney, where copolymers are excreted. The HPMA copolymer is thus a promising strategy for in vivo delivery of Bcl-2-targeting compounds to solve their poor solubility problem and to enhance tumor selectivity.

Keywords: apoptosis, HPMA copolymer bioconjugate, HA14-1, xenograft



123-132

Epstein-Barr Virus downregulates expression of DNA-Double strand break repair proteins in nasopharyngeal cancer

Author(s): Dr. Prabha Balaram,

Abstract: Nasopharyngeal carcinoma (NPC) is a unique cancer due to its etiology, and incidence and viral infection pattern. Radiotherapy (RT) is the main treatment modality and these lesions vary considerably in treatment response. In the present study, the effect of EBV and HPV infection on response to treatment based on the expression of DNA-repair proteins, ATM and DNA-PKcs was assessed in NPC based on the rationale that the expression of these proteins play important roles in the response to RT in NPC. 103 NPC biopsies and 26 benign adenoid lesions of the Nasopharynx were collected before treatment and graded according to WHO classification. Response to treatment(radiation) was evaluated clinically. Western blotting and immuno histochemical analysis were used for evaluating the expression of DNA-PKcs and ATM and PCR using specific primers was used for detection of EBV and HPV. EBV presence was also confirmed by EBER-ISH. 63% of NPC were EBV+ve and 30% HPV+ve in the samples studied. Expression of ATM and DNA-PKcs was significantly increased in NPC when compared to benign samples (p<0.001). However, NPC showing EBV infection showed downregulation of ATM and DNA-PKcs expression (p=0.009 and p=0.011), in comparison to HPV infected NPC’s (p=0.001 and p=0.003). NPC patients with HPV positivity also showed a significantly poor response to treatment (p=0.002)with a higher rate of recurrence and lower disease free survival. The study indicates that EBV infection down regulates the expression of DNA-repair proteins and renders NPC sensitive to RT while HPV infection up regulates their expression making the tumors resistant to therapy. The results of the study also indicate that assessment of expression of DNA-PKcs and ATM in biopsy specimens can be used as criteria to identify radio-resistant NPC’s and selection of appropriate therapy regimens.

Keywords: DNA-PKcs, ATM, Nasopharyngeal carcinoma, EBV, HPV, Radiotherapy, DNA-double-strand break (DSB) repair proteins.



133-146

Mechanisms of malignant glioma immune resistance and sources of immunosuppression

Author(s): Dr. Carol A. Kruse,

Abstract: improve survival in this select population of cancer patients. Gene Therapy and Molecular Biology Vol 10, page 133 High grade malignant gliomas are genetically unstable, heterogeneous and highly infiltrative; all characteristics that lend glioma cells superior advantages in resisting conventional therapies. Unfortunately, the median survival time for patients with glioblastoma multiforme remains discouraging at 12-15 months from diagnosis. Neuroimmunologists/oncologists have focused their research efforts to harness the power of the immune system to improve brain tumor patient survival. In the past 30 years, small numbers of patients have been enrolled in a plethora of experimental immunotherapy Phase I and II trials. Some remarkable anecdotal responses to immune therapy are evident. Yet, the reasons for the mixed responses remain an enigma. The inability of the devised immunotherapies to consistently increase survival may be due, in part, to intrinsically-resistant glioma cells. It is also probable that the tumor compartment of the tumor-bearing host has mechanisms or produces factors that promote tumor tolerance and immune suppression. Finally, with adoptive immunotherapy of ex vivo activated effector cell preparations, the existence of suppressor T cells within them theoretically may contribute to immunotherapeutic failure. In this review, we will summarize our own studies with immunotherapy resistant glioma cell models, as well as cover other examined immunosuppressive factors in the tumor microenvironment and immune effector cell suppressor populations that may contribute to the overall immune suppression. An in-depth understanding of the obstacles will be necessary to appropriately develop strategies to overcome the resistance and

Keywords: T regulatory cells, cellular immunotherapy, immunotherapy, brain tumors, CTL resistance, effector T lymphocytes, FasL, immunosuppressive mechanisms, apoptosis, adhesion molecules, extracellular matrix proteins, cytokines



147-160

Characterization of the cytotoxic effect of a chimeric restriction enzyme, H1o-FokI

Author(s): Dr. Donald B. Sittman,

Abstract: Our primary goal was to create an efficient cytotoxic agent. To do this, we created a gene that expresses a chimeric hybrid of the linker histone, H10 and the nuclease domain of the type IIs restriction enzyme, FokI. The linkage of the FokI nuclease domain to a high affinity but low DNA-sequence-specificity binding protein is unique. It is highly cytotoxic. We demonstrate, by transiently transfecting 3T3 mouse fibroblasts, that 63% of the cells taking up the chimeric gene are killed. The chimeric protein is localized to the nucleus. An extract of the protein produced in E. coli degrades DNA, indicating that it is nucleolytically active. The ultimate mechanism through which the chimeric protein produces cell death is likely through the induction of apoptosis.

Keywords: Chimeric nuclease, cytotoxic, apoptosis



161-164

The study of 16S rRNA in meningitis by molecular biology assay

Author(s): Dr. Hossein Goudarzi,

Abstract: In order to treatment of patients with meningitis rapid diagnosis of agent is very important. Now all of researchers have approved qualification and efficiency of molecular tests. Detection of bacteria from cerebrospinal fluid (CSF) and blood is big cumbersome as atmosphere condition and usage of antibiotics by patients. We explored on CSF samples by PCR test and used DG74 and RDR80 primers for 16S rDNA sequence. Our cases are children with meningitis symptoms that had referred to hospitals at Tehran. This samples are different from culture, cell counter and protein glucose amounts. After researching we reached to these results that 23.5% of case were positive as bacterial culture and 41.1% of them were positive as PCR test. So sensitivity of PCR was95.23%, specificity of PCR was 96.66% and efficiency of PCR was 96%.

Keywords: 16srRNA, meningitis



165-172

FLT3-ITD: technical approach and characterization of cases with double duplications

Author(s): Dr. Emanuela Frascella,

Abstract: FLT3-Internal Tandem Duplication (ITD) of the juxtamembrane domain is one of the most common genetic alterations in acute myeloid leukemia (AML) and in some FAB subgroups seems to represent an unfavorable prognostic factor. Thus, its correct identification is critical. We analyzed 261 AML cases to individuate FLT3-ITD by RT-PCR and we compare different techniques (agarose and polyacrilamide gel electrophoresis, sequence and Genescan of PCR products) to define FLT3-ITD presence, length and number. All 53 positive cases were identified by electrophoresis on agarose gel. The sequence of the FLT3-ITD amplicons eluted from polyacrilamide gel was successfully performed while failing from agarose gel. We compared different methods of purifying PCR products from polyacrilamide gel to identify the fastest and most effective one. Genescan analysis was used to confirm the presence and the length of the ITD and to study the rate between ITD/WT transcripts. In our experience electrophoresis on 2% agarose gel is adequate for identifying FLT3-ITD, while purification from polyacrilamide gel is suggested for sequencing. In our series we found 20% of positive cases, 7.5% of these lacked FLT3 wild-type transcript and 13.2% showed two different FLT3-ITDs. In addition we identify 2 cases carrying 2 FLT3-ITD with the same length but different nucleotide sequence.

Keywords: FLT3-ITD, AML, acrylamide, purification, mutant level



173-178

The Human VG5Q Gene Transcript is Over Expressed in Colorectal and Bladder Carcinomas

Author(s): Dr. Imad J. Matouk,

Abstract: We studied the pattern of the human VG5Q (AGGF1) mRNA expression in both normal and noeplastic colorectal and bladder tissues. VG5Q mRNA was detected by RT-PCR technique. VG5Q is weekly expressed in the majority of normal cases (n=12). Seven of eight colorectal carcinomas (87.5%) overexpressed VG5Q mRNA when compared to their corresponding normal colorectal tissues of the same patient. The level of VG5Q expression in primary tumor is also upregulated in (75%) of the cases when compared to their corresponding liver metastasis. No consistent relationship in the expression level of VG5Q could be deduced when comparing normal colorectal samples to their liver metastasis colorectal tumors. Comparing 4 normal bladder and 16 bladder carcinomas samples reveal that VG5Q expression is also upregulated in bladder carcinomas. The level of VG5Q expression is more frequently upregulated in low grade when compared to high grade bladder carcinomas. These are the first results indicating the association of the newly discovered VG5Q gene transcript with human colorectal and bladder carcinomas. Further studies are needed to evaluate the usage of VG5Q as a complementary histopathologic and a candidate tumor marker among other modalities in both and other types of cancers.

Keywords: Colorectal and bladder carcinomas; VG5Q; Tumor marker; Cancer grade; Primary and secondary growth



179-184

Title-loss of βcatenin is an independent prognostic factor in ovarian carcinomas: A multivariate analysis

Author(s): Dr. Cristina Faleiro-Rodrigues,

Abstract: In ovarian carcinomas, numerous studies have shown consistent prognostic significance of FIGO stage and residual tumour as independent prognostic factors. However, these prognostic factors alone cannot accurately predict disease outcome since a considerable degree of heterogeneity remains within the various subgroups limiting the predictive value of these factors. Therefore, the identification of new molecular markers that may possibly distinguish patients at a higher risk is of great importance. In two previous studies, the individual loss of E-cadherin and the individual loss of β-catenin were important prognostic factors of poorer overall survival in patients with ovarian carcinomas. Purpose of the present study was to re-analyse the immunohistochemical expression of E- cadherin and β-catenin in 104 patients with ovarian carcinomas, and evaluate whether these two proteins continue to be important independent prognostic factors when assessed together in a multivariate Cox ́s proportional hazard regression analysis. Results In the multivariate analysis, the most important independent prognostic factors of poorer overall survival were loss of β-catenin expression ([HR], 5.79, 95% CI, 2.38 to 14.10; P=0.0001), FIGO stage IV ([HR], 7.19, 95% CI, 1.02 to 50.8; P=0.04) and residual tumour ([HR], 6.78, 95% CI, 1.41 to 32.56; P=0.034). Conclusion The loss of β-catenin expression is a stronger prognostic factor than E-cadherin. The findings in the present study and previously reported data suggest that β-catenin is a significant prognostic indicator in patients with epithelial ovarian cancer, however, these results should be supported by more and larger studies.

Keywords: ovarian cancer, cell adhesion, epithelial cadherin, β-catenin, immunohistochemistry



185-192

New generations of retroviral vector for safe, efficient and targeted gene therapy

Author(s): Dr. Walter H. Günzburg,

Abstract: Retroviral vectors were the first virus vectors to be used in gene therapy trials and have proved to be successful for the treatment of X-linked severe combined immunodeficiency. However, there are safety concerns associated with the use of retroviral vectors or indeed delivery systems based upon viruses in general. Over the last few years, we have been developing retroviral vectors with the aim of (i) removing the retroviral promoter in transduced cells (ii) obtaining limited expression of therapeutic genes in therapeutically relevant cells by the inclusion of targeting promoters in place of the retroviral promoter (iii) being able to stably produce retroviral vectors carrying toxic genes from cells. Two of these vector systems, promoter conversion vectors and reconstituting vectors, have been described in proof of principle studies, but suffered from reduced titres that precluded their effective use in the clinic. A number of vector optimisation modifications have been made to these vectors, resulting in the successful improvement of both titre and expression levels such that these vectors are now suitable for use in clinical trials. The use of such optimised vectors for in vitro and in vivo applications using a number of different genes of interest will be described. Future successful gene therapy of solid tumours may require the use of replicating vectors. The application of many of the principles learned from the vector optimisation modifications described above to replicating MLV and MMTV based vectors will be described along with data demonstrating efficient tissue specific expression targeting.

Keywords: retroviral vector, murine leukaemia virus, mouse mammary tumour virus, improvements, promoter conversion vector, reconstituting vector



193-198

The association of endothelial constitutive Nitric Oxide Synthase polymorphisms with family history of coronary heart disease in men

Author(s): Dr. Nasser M. Al-Daghri,

Abstract: It has been reported that endothelial nitric oxide synthase (ecNOS) gene polymorphism is associated with the risk of CHD, acute myocardial infarction (AMI) and atherosclerosis but hitherto no subjects with a family history of CHD have been examined. 292 native Saudi males of matching ages were drawn from normal, healthy male volunteers attending the blood bank at Alshmasee and the King Khalid University Hospital in Riyadh, Saudi Arabia. Blood samples were collected for the determination of lipids profiles using routine laboratory methods and Genotype was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. The genotype frequencies for bb, ab and aa were 31.5, 53 and 5.5% respectively and the calculated allele frequencies for the ecNOS4b (0.65) and ecNOS4a (0.35) were not statistically different. The subjects were divided according to the family history of CHD, with an excess of individuals homozygous for bb and aa among the subjects who have a history of CHD standing at 61% and 12%, compared with those who do not have a history of CHD (59% and 4% respectively, p= 0.04). The ecNOS gene was found to be associated with family history of Coronary heart disease in Saudis male subjects more attention to these patients should be considered.

Keywords: ecNOS gene, History of coronary heart disease, Physical activity, Type 2 Diabetes



199-206

Apoptotic signaling induced by Tiazofurin-an in vitro study

Author(s): Dr. Neeta Singh,,

Abstract: pathway. Gene Therapy and Molecular Biology Vol 10, page 199 Tiazofurin (TR), is a novel anticancer agent exhibiting potent cytotoxic activity in malignant cell lines. It exhibits at least two different mechanisms of action. First is by inhibition of inosine 5’ monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme for guanylate (GTP, dGTP) biosynthesis and second is by the induction of apoptosis. But the mechanism of induction of apoptosis is not clear. The purpose of the present study was to elucidate the apoptotic signaling induced by TR in different human cancer cell lines. The effect of TR was studied on SiHa (human cervical cancer cell line), Hep2 (human laryngeal cancer cell line) and Ca Ski (human cervical cancer cell line) cells. Morphological examination, flowcytometry and Caspase-3 assay were used for detection of apoptosis. Expression of various proteins was seen by Western blotting. Our results reveal that TR at a dose of 100μM induces apoptosis in SiHa and Hep2 cells whereas for Ca Ski cells this dose is 150μM as studied by morphology and flow cytometry. A downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed whereas the expression level of the pro-apoptotic protein Bax remained unaffected in all these cell lines. An upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF). A moderate increase in caspase-9 activity was seen. There was a significant increase in caspase-3 activity, which was accompanied by PARP cleavage. Release of cytochrome c from the mitochondria to the cytosol was also observed. The findings suggest that TR induces apoptosis in SiHa, Hep2 and Ca Ski cells via the intrinsic mitochondrial

Keywords: Tiazofurin, apoptosis, mitochondria, cytochrome



207-222

Effects of spatial configuration on tumor cells transgene expression

Author(s): Dr. Liliana M. E. Finocchiaro,

Abstract: We investigated the impact of the multicellular architecture on transgene expression of LM05e and LM3 spontaneous Balb/c-mammary adenocarcinoma and HEp-2 human laryngeal squamous carcinoma cell lines. When transferred from monolayers to spheroids, tumor cells strongly enhanced transient transgene expression, which surprisingly was still detectable 75 days after lipofection. The cytomegalovirus immediate early promoter (CMVie) yielded a very high β-galactosidase (βgal) transgene expression, which resulted 8-, 6- and 3-fold greater in LM05e, LM3 and HEp-2 spheroids than the corresponding monolayers. The SV40 early promoter displayed both, a lower spheroids/monolayers ratio and about 10% of βgal expression driven by CMVie. Cis-addition of Epstein Barr virus EBNA-1/oriP cassette enhanced the CMVie-driven transgene expression only in human HEp-2. Deletion of a 325 bp 5’ fragment of the CMVie promoter dropped spheroids βgal expression to 5%. This effect was restored to 10-25% or 25-60% by the insertion of one KCS (18 bp) or four myc-max consensus sequences (67 bp) . When spheroids disassembled and grew as monolayers, βgal activity dropped accordingly

Keywords: multicellular tumor spheroids, persistent gene expression, non-viral vectors



223-232

Use of lectin as an anchoring agent for adenovirus- microbead conjugates: Application to the transduction of the inflamed colon in mice

Author(s): Dr. Takeshi Sano,

Abstract: the therapy of IBD. Gene Therapy and Molecular Biology Vol 10, page 223 Virus-mediated delivery of therapeutic transgenes to the inflamed colon offers a great potential to serve as an effective therapeutic strategy for inflammatory bowel disease (IBD). However, the transduction of the inflamed colon by viral vectors upon intra-colonical administration is generally poor, primarily because of the inability of administered viral vectors to associate stably with the colonic tissue. We investigated if the use of adenoviral vectors in the form of virus-microbead conjugates could enhance the transduction efficiency of the inflamed colon. In particular, a lectin, concanavalin A (Con A), was tested as an anchoring agent for adenovirus-microbead conjugates. The co-attachment of Con A allowed adenovirus-microbead conjugates to associate stably with target cells when analyzed in vitro. Intra-colonical administration of adenovirus-microbead conjugates containing Con A resulted in efficient transduction of the inflamed colon, while little transduction was seen with adenovirus- microbead conjugates without Con A or free adenoviral vectors. When adenoviral vectors carrying the mouse interleukin-10 gene were used, local interleukin-10 levels became considerably higher upon intra-colonical administration of adenovirus-microbead conjugates containing Con A. These results demonstrate that Con A can serve as an effective anchoring agent for adenovirus-microbead conjugates and suggest that adenovirus-microbead conjugates containing Con A may be useful for efficient delivery of therapeutic transgenes to the inflamed colon for

Keywords: Adenoviral vectors; virus-microbead conjugates; lectin; interleukin-10; inflammatory bowel disease



233-244

Replicating minicircles: Generation of nonviral episomes for the efficient modification of dividing cells

Author(s): Dr. Juergen Bode,

Abstract: nuclear matrix and its authentic segregation. Gene Therapy and Molecular Biology Vol 10, page 233 Nonviral replicating circular episomes are a rather new addition to the field of mammalian expression vectors. After their establishment, which conventionally requires an initial phase under selection pressure, these entities utilize the replication apparatus of the host cell to replicate in accord with the cell cycle. The requirements of a selection agent, the gradual inactivation by cellular defense mechanisms, and a limited cloning capacity (up to 5 kb could be realized for the prototype) have remained the critical parameters. Here we introduce a site-specific recombination-based strategy that permits the excision of prokaryotic vector parts after the parental construct has been amplified as a plasmid. The remaining 4 kb ́minicircle ́ consists of only one active transcription unit and a scaffold/matrix attachment region (S/MAR). In contrast to the parent plasmid vector it can be established in the absence of selection, it is not subject to epigenetic silencing and it replicates stably without a sign of integration. In further contrast to available minicircles that are maintained only in non-dividing tissues our minicircle represents the first example that is suited for the modification of dividing cells and tissues due to its association with the

Keywords: replicating episome, nonviral vector, minicircle, S/MAR, maintenance element, segregation



245-250

Cloning, Expression and Purification of a novel anti- angiogenic factor-Tumstatin

Author(s): Dr. Chongbi Li,

Abstract: blot identified it’s right. Gene Therapy and Molecular Biology Vol 10, page 245 Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. Tumstatin peptide is an angiogenesis inhibitor derived from type IV collagen and inhibits in vivo neovascularization induced by vascular endothelial growth factor (VEGF), Here, we firstly showed the expression, cloning and purification of tumstatin from Chinese abortus kidney tissue by RT-PCR, and the construction of pET-His expressive plasmid in prokaryotic cells. Also its’ activity was examined by mouse antiserum against native Tumstatin. The results indicated E.coli BL21(DE3)plysS/ pET-His-tumstatin was induced 3 h by 0.2 mmol/L IPTG at 30°C, and got a high-level expression of 37.9%. The Tumstatin protein was one-step purified by immobilized metal-chelating affinity chromatography (IMAC) and its purity was above 95%. Western

Keywords: tumstatin, cloning and expression; IMAC



251-254

Plasmodium and host carbonic anhydrase: molecular function and biological process

Author(s): Dr. Viroj Wiwanitkit,

Abstract: Carbonic anhydrase (CA) is an enzyme that catalyzes an interconversion of CO2 and HCO3-. CA is present at high levels in humans and Plasmodium spp. However, the function of CA in malarial infection is not well characterized. Here, the author used a new gene ontology technology to predict molecular function and biological process of CA. Using GoFigure server, the molecular function and biological process of human and P. falciparum CA are predicted. Comparing to human CA, the P. falciparum CA has similar molecular functions as carbonate dehydratase activity and zinc ion binding. Although the basic sequences for human and P. falciparum CA are totally different, the molecular functions are similar. This finding implies that any treatment aiming at blocking the functions of P. falciparum CA can affect human CA. Thus any drug targeting at P. falciparum CA might not be a magic bullet. The more specific structural antagonist that can directly block amino acid of P. falciparum CA is more

Keywords: human, Plasmodium falciparum, carbonic anhydrase, function



255-262

Isolation of genes controlling apoptosis through their effects on cell survival

Author(s): Dr. Gwyn T. Williams,

Abstract: The identification of the most suitable molecular targets for gene and drug therapy is the crucial first step in the development of new disease treatments. The rational identification of such targets depends on a detailed understanding of the pathological changes occuring at the molecular level. We have applied forward genetics approaches to the identification of the critical genes involved in the control of apoptosis in mammalian cells, since defective control of apoptosis underlies many diseases, including cancer and neurodegenerative diseases. We have identified two groups of genes by their effects on cell survival using retroviral cDNA functional expression cloning and retroviral insertional mutagenesis. The identification of these novel genes opens up new areas for apoptosis research and subsequently for the development of new gene and drug therapies.

Keywords: apoptosis, forward genetics, functional cloning, retroviral insertional mutagenesis, oncogenes, tumour suppressor genes



263-268

The prevalence of antibiotic resistance in anaerobic bacteria isolated from patients with skin infections

Author(s): Dr. Gita Eslami,

Abstract: Antibiotic resistance in Anaerobic bacteria and the lack of proper outline to treatment of anaerobic infections have been increased in recent years, In this study 100 patients with skin infections (10-60 years old) were considered. Specimens were collected in the sterile condition and transported and cultured in the Thioglycolate media. After growing and staining of bacteria (gram staining) from selective media, bacteria were cultured in the differentiated media. Strains that were isolated, undergone antibiogram test (Kirby bauer method). Skin infections are usually polymicrobial involving aerobic and anaerobic bacteria. Common aerobic and anaerobic facultative bacteria contained: Staphylococcus aureus (37.3%), non coagolase Staphylococci (8.5 %), group A streptococci (16.3 %), group D enterococci (5.7%), E.coli (15.6 %), enterobacter-spp (5.6%), citrobacter-spp (0.8%), Pseudomonas aeruginosa (6.9%), proteus-spp (2.7%), others (0.6%). Predominant anaerobic bacteria contained: Peptostreptococcus-spp (42.5%), pigmented prevotella and Porphyromon-spp (5.4%), Fusobacterium (7.6%) Bacteroides-spp (23.2%), Clostridium-spp (18.4%), Propionebacteriom acnes (2.1%), others (0.8%). Atibiogram test was done on aerobic-anaerobic facultative bacteria. Susceptibility of these bacteria were as following: Cefizoxim100%, Ciprofloxcin 98%, Ceftazidim 82%, Tobramycin 47%, and Amikacin 33%. And their resistance to Gentamycin was 97%, Penicillin 93%, Cloxacillin 86%, and Erythromycin 62%. In anaerobic bacteria, susceptibility to Ciprofloxacin was 100%, Ceftyzoxim 100, Ceftazidim 91% Rifampin 76%, Colistin 67%, and their resistance to Penicillin was 95%, Erythromycin 83%, Cloxacillin 85%. Susceptibility of both anaerobic and aerobic bacteria to Ceftizoxim was 100 %, so we suggest this drug for treatment of many skin infections.

Keywords: Antibiotic resistance, anaerobic bacteria, skin infection



269-276

Transfection of the anti-apoptotic gene bcl-2 inhibits oxidative stress-induced cell injuries through delaying of NF-κB activation

Author(s): Dr. Nobuhiko Miwa,

Abstract: We investigated the relation between endogenous NF-κB and exogenous overexpressed Bcl-2 in rat fibroblastic cells (Rat-1) in response to H2O2 after confirming the cytoprotective effect of Bcl-2 against oxidative stresses such as in vitro treatment with H2O2 and in vivo hepatic post-ischemic reperfusional (I/R) injury. Exogenous Bcl-2, which was expressed by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of human bcl-2 that was incorporated into an SV2 vector, prevented I/R-induced hepatic injuries such as cellular DNA strand cleavages more effectively than the non-transfection treatment. The bcl-2-transfected Rat-1 fibroblasts exerted the cytoprotective effect against H2O2 of 50-250 uM more markedly than the SV2 vector- transfected or non-transfected counterpart cells. Immunocytochemical analysis and electrophoretic mobility shift assay (EMSA) showed that intracellular activation of NF-κB in bcl-2-transfectants was repressed more appreciably than in SV2-transfectants at a period as early as 30 min after H2O2 stimulation, but, at advanced periods of 90 and 120 min, was increasingly exhibited up to the similar and exceeding levels relative to those of SV2-transfetants, respectively. Thus the prevention by the anti-apoptotic gene bcl-2 against oxidative stress-induced injury may be attributed at least partly to the repressed early activation and/or the delayed activation of NF-κB. The results provide the foundation for redox-mediated gene therapies using bcl-2 gene directing at ameliorative effects against

Keywords: Bcl-2, NF-κB, H2O2, oxidative stress, apoptosis




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