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PubMed2001

Bidirectionalization of polar promoters in plants.

A typical eukaryotic promoter consists of a minimal promoter and other upstream cis elements. The minimal promoter is essentially a TATA box region where RNA polymerase II, TATA-binding protein (TBP), and TBP-associated factors (TAFs) bind to initiate transcription, but minimal promoters alone have no transcriptional activity. The cis elements, to which tissue-specific or development-specific transcription factors bind, individually or in combination, determine the spatio-temporal expression pattern of a promoter at the transcriptional level. The arrangement of upstream cis elements followed by a minimal promoter sets the polarity of the promoter. Promoters in plants that have been cloned and widely used for both basic research and biotechnological application are generally unidirectional, directing only one gene that has been fused at its 3' end (downstream). It is often necessary to introduce multiple genes into plants for metabolic engineering and trait stacking. It is also desirable to minimize or avoid repeated use of a single promoter that may cause transcriptional gene silencing. Here we describe a strategy to make polar promoters bidirectional so that one promoter can direct the expression of two genes, one on each end of the promoter.

Arabidopsis↗
PubMed1986

Correction of adenosine deaminase deficiency in cultured human T and B cells by retrovirus-mediated gene transfer.

A retroviral vector called SAX, containing the cloned human cDNA for adenosine deaminase (ADA), has been constructed and used to introduce the ADA gene into cultured T- and B-lymphocyte lines derived from patients with ADA deficiency. DNA analysis showed that the SAX vector was inserted intact into the T and B cells at approximately one copy per cell. The treated cells produced the characteristic isozymes of human ADA at a level similar to normal T and B lymphocytes. It is known that ADA-deficient lymphocytes are unusually sensitive to high levels of 2'-deoxyadenosine, and this is the mechanism thought to underlie the selective lymphocytotoxicity associated with ADA deficiency in vivo. Expression of the introduced ADA gene was sufficient to reverse the hypersensitivity of these genetically deficient lymphocytes to 2'-deoxyadenosine toxicity. These results support the suggestion that retroviral vector gene-delivery systems show promise for application to human gene therapy.

Adenosine Deaminase↗
PubMedDate not supplied

Rapid isolation, expansion, and differentiation of osteoprogenitors from full-term umbilical cord blood.

There is an urgent clinical requirement for appropriate bone substitutes that can be used for the repair and regeneration of diseased or damaged skeletal tissues. Cell-sourcing limitations in particular have affected progress, largely because of the shortage of accessible tissues capable of yielding sufficient numbers of viable osteoprogenitor cells. Previous work has suggested that umbilical cord blood (UCB) contains circulating progenitor cells (mesenchymal stem cells) capable of osteogenic differentiation, although a comparable number of reports refute this claim. From a screen of more than 20 different culture conditions, we have identified an optimal, simple, and reliable technique to generate, from full-term human UCB, stromal cells with the ability to undergo rapid osteogenic differentiation. By comparing different sorting and culture strategies, we demonstrated that early exposure of mononuclear UCB cells to medium conditioned by osteoblastic cells in the presence of osteogenic supplements and human plasma, markedly increased the frequency of stromal cell growth, the rate of osteogenic differentiation, and their attachment to and spreading on calcium phosphate scaffolds. These findings suggest that full-term UCB may act as an appropriate source of osteoprogenitor cells, which will impact significantly on the development of autologous tissue- engineered bone constructs.

Acid Phosphatase↗
PubMed2006

Making better influenza virus vaccines?

Killed and live influenza virus vaccines are effective in preventing and curbing the spread of disease, but new technologies such as reverse genetics could be used to improve them and to shorten the lengthy process of preparing vaccine seed viruses. By taking advantage of these new technologies, we could develop live vaccines that would be safe, cross-protective against variant strains, and require less virus per dose than conventional vaccines. Furthermore, pandemic vaccines against highly virulent strains such as the H5N1 virus can only be generated by reverse genetics techniques. Other technologic breakthroughs should result in effective adjuvants for use with killed and live vaccines, increasing the number of available doses. Finally, universal influenza virus vaccines seem to be within reach. These new strategies will be successful if they are supported by regulatory agencies and if a robust market for influenza virus vaccines against inter-pandemic and pandemic threats is made and sustained.

Adjuvants, Immunologic↗
PubMed1987

Potential oncogenic hazards posed by oncogene encoded proteins.

The theoretical risk from oncogene encoded proteins is limited to growth factors, which may be secreted by cells in which biologically derived products might be manufactured. Because these peptides do not replicate, their effect is finite. In addition, their effects are reversible. Growth factors do not ordinarily appear to be oncogenic. Even under circumstances in which they contribute to oncogenicity, repeated administration of high concentrations of growth factors (several micrograms per kilogram) would appear to be required for them to serve as co-factors in the carcinogenic process, and the resulting tumors appear to remain dependent upon the continued presence of the growth factor for continued tumor growth.

Animals↗
PubMed1996

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

Animals↗
PubMed2000

Implantation of rat insulinoma cell line into cyclosporine treated rats. Effect of the in vivo environment on beta-cell specific gene expression.

BACKGROUND: Transplantation of engineered beta cell-derived lines is a promising modality for cell-based therapy of diabetes mellitus. The in vivo environment and antirejection and other medications may have significant effects on the differentiation and proliferation of the transplanted beta cells, thus affecting their function. The effect of the in vivo environment on expression of genes encoding proteins involved in insulin production, secretion, and glucose sensing were analyzed in the RIN 104638 cell line. METHODS: RIN 104638 cells, were used for s.c. implantation in cyclosporine treated rats and for parallel in vitro culture. The differential expression of the insulin, PDX-1, GLUT-2, and glucokinase genes were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: The in vivo environment of cyclosporine-treated rats, preserved most of the differentiated characteristics of the implanted cells. Insulin and glucokinase gene expression were maintained at high levels, although GLUT-2 expression decreased. This was in contrast to the substantial decrease of all the three genes expression when cultured in vitro. Cyclosporine treatment reduced insulin and GLUT-2 gene expression in in vitro culture. CONCLUSIONS: Beta cell implantation in cyclosporine-treated rats induces alteration in expression of genes pivotal to insulin production and secretion and the glucose sensing abilities. The normal in vivo environment improves the implanted b cell function by increasing the insulin gene expression and content. Furthermore, it reverses some of the dedifferentiating changes caused by the in vitro culture. This may have a positive effect on the therapeutic efficiency of this cell line.

Actins↗
PubMed2005

[Expression of mineral-associated markers in human dental follicle cells in vitro].

OBJECTIVE: To culture human dental follicle cells in vitro and observe their mineralization characteristics. METHODS: Human dental follicle tissues were digested with bacterial collagenase and cultured to obtain dental follicle cells. Mineralization characteristics of these cells were identified by immunohistochemistry and reverse transcription (RT)-PCR. Dental follicle cells were incubated with mineralization-inducing agents to observe the formation of mineralized nodules. RESULTS: Immunohistochemistry staining revealed expressions of type I collagen, type III collagen, osteopontin and osteonectin in the cytoplasm of dental follicle cells cultured in vitro. Low levels of bone sialoprotein, osteocalcin and alkaline phosphatase mRNA were also detected by RT-PCR. Mineralized nodules were observed after 20 days of incubation with mineralization-inducing agents with positivity for von-Kossa staining. CONCLUSION: Human dental follicle cells cultured in vitro show some features of osteoblasts or cementoblasts and can be used as the seed cells for periodontal tissue engineering.

Calcification, Physiologic↗
PubMed1994

d-Limonene induced hyaline droplet nephropathy in alpha 2u-globulin transgenic mice.

d-Limonene is a hyaline droplet inducing agent and produces nephrotoxicity in male rats when the 1,2-epoxide metabolite binds to alpha 2u-globulin. Mice, which do not synthesize alpha 2u-globulin, are resistant to hyaline droplet nephropathy. In this study, the ability of d-limonene to cause hyaline droplet nephropathy in a transgenic mouse engineered to express alpha 2u-globulin was evaluated. The C57BL/6-derived mice excreted 0.4 +/- 0.1 mg alpha 2u-globulin/day, or approximately 16 mg alpha 2u-globulin/kg body wt. This represents about 30% of the amount excreted by adult male rats (11.9 +/- 1.1 mg/day or approximately 48 mg/kg). Transgenic mice excreted less mouse urinary protein (9.3 +/- 1.2 mg/day) than normal mice (15.1 +/- 1.6 mg/day). Unlike normal male rats, untreated transgenic mice did not show significant spontaneous hyaline droplet formation. Liver microsomes from naive transgenic mice oxidized d-limonene to the cis- and transisomers of the 1,2-epoxide, and following oral treatment with [14C]d-limonene reversible binding of d-limonene equivalents to renal cytosolic proteins was observed. Furthermore, with d-limonene treatment, hyaline droplets were observed in the transgenic mouse kidneys. These droplets, however, were much smaller in size than those seen in d-limonene-treated male rats. The accumulation of alpha 2u-globulin in the kidneys of transgenic mice and normal male rats before and after d-limonene treatment was analyzed by Western blotting. These results indicated that alpha 2u-globulin was present in the kidneys of the control transgenic mice, despite the lack of spontaneous hyaline droplet formation. After d-limonene treatment, approximately a three-fold increase in alpha 2u-globulin in the transgenic mouse kidney was observed, a response similar in magnitude to that seen in d-limonene-treated male rats. These results indicate that expression of alpha 2u-globulin in a species that does not normally develop hyaline droplet nephropathy is necessary and sufficient to render that species sensitive to this renal toxicity.

Alpha-Globulins↗
PubMed2003

Assessment of retroviral activity using a universal retrovirus chip.

A DNA chip-based assay is described for parallel detection and identification of a wide variety of human and mammalian exogenous and endogenous retroviruses. The assay combines multiplex polymerase chain reaction (PCR) using fluorochrome-modified primer mixtures and chip hybridization. The microarray is composed of retrovirus-specific synthetic oligonucleotides as capture probes deposited on glass slides. The retrovirus chip can be used to assess the occurrence of reverse transcriptase (RT)-related transcripts in biological samples of human and mammalian origin. For example, distinct expression profiles of human endogenous retroviruses (HERV) were established reproducibly in human white blood cells, mammary gland and other human tissues. In particles released by human cells, packaging of specific HERV transcripts could be observed. Monitoring of human exogenous retroviruses (HIV, HTLV) and detection of putative cross-species transmissions (MLV, PERV) in human samples was efficient and reliable. The DNA chip should be an excellent tool for the detection of most relevant retroviruses and offers insights into differential retroviral activities and replication strategies. Furthermore, it could improve significantly the safety of gene therapy, tissue engineering, xenotransplantation and production of therapeutic polypeptides in cell culture.

Animals↗
PubMed2005

Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors.

Wheat alpha-amylase inhibitors represent an important tool in engineering crop plants against bean bruchids. Because Acanthoscelides obtectus is a devastating storage bean insect-pest, we attempted to purify and characterize its gut alpha-amylases, to study their interaction with active proteinaceous inhibitors. Two digestives alpha-amylases (AoA1 and AoA2) were purified from gut larvae, showing molecular masses of 30 and 45 kDa for each one, respectively. The stoichiometry interaction between these alpha-amylases with two wheat inhibitors (0.19 and 0.53) showed a binding complex of 1:1 enzyme:inhibitor. In vivo activities of these inhibitors against A. obtectus were also evaluated using a rich ammonium sulfate inhibitor fraction (F(20)(-)(40)) and purified inhibitors after reversed phase high-performance liquid chromatography columns. Incorporation of three different inhibitor concentrations (0.25, 0.5, and 1.0% w/w) into artificial seeds showed that addition of the purified 0.19 inhibitor at the highest concentration (1.0%) reduced the larval weight by 80%. Similar data were observed when 0.53 inhibitor was incorporated at 0.5%. When the concentration of purified 0.53 was enhanced to 1.0%, no larvae or adult emergence were observed. Our data suggest that these alpha-amylase inhibitors present great potential for use in Phaseolus genetic improvement programs.

Animals↗
PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗
PubMed1998

[The current status of research on a cholera vaccine].

Cholera remains today a major health problem in most developing countries. The long-term control of cholera depends on the improvement of hygiene but this is a distant goal for many countries. The availability of an effective cholera vaccine is thus important for the prevention of cholera in such countries. More than a century after the first attempt to vaccinate against cholera by Ferran in Spain, there is still no truly effective cholera vaccine. A bacterial fraction vaccine, referred to as CH1 +2 was prepared by Professor A. Dodin. A field trial of this vaccine was carried out in Zaire in 1983. Significant protection was observed but this vaccine was not evaluated in additional trials. Two other oral cholera vaccines, developed in Sweden and in the USA, were widely experimented on human beings: a combination of cholera toxin B-subunit and inactivated bacterial cells, and a live attenuated vaccine containing the genetically manipulated Vibrio cholerae O1 strain CVD 103-HgR. Despite their efficiency as evaluated in field trials (inactivated vaccine) or on volunteers (live vaccine), these vaccines have drawbacks that may limit their usefulness as practical vaccines. Protection induced by the inactivated vaccine was transient in young children, lasting only approximately for six months. One of the safety concerns associated with live vaccines is a possible reversion to virulence. Efforts should be continued to find a better cholera vaccine. A new vaccine development program based upon the hypothesis that immunoglobulin G directed to the O-specific polysaccharide of Vibrio cholerae O1 could confer protective immunity to cholera. This program may lead to the development of a cholera conjugate vaccine to elicit protection in infants.

Adjuvants, Immunologic↗
PubMed1994

Pancreatic islet cell transplant.

It is obvious that islet transplantation as an isograft can produce normoglycemia and can prevent and reverse early complications of diabetes in rodents. It is possible to produce normoglycemia in immunosuppressed diabetic patients with human islet transplants. It is possible to prevent rejection of islets in animals without continuous immunosuppression by immunoalteration of the islets, and this approach will be attempted in human diabetes. Finally, the encapsulation approach for prevention of rejection of islets is promising. Large animals will be used to test prototypes of the hollow fibers for humans, and if these experiments are successful, it will be possible to progress rapidly to human diabetes. The first approach with encapsulation in human diabetes would be to use human islets, then encapsulated pig islets, and finally genetically engineered beta cell lines when they become available. I hope that these approaches in islet transplantation will prevent the devastating diabetic complications that Henry Dolger demonstrated many years ago could occur in diabetic patients even though those patients are taking insulin.

Animals↗
PubMed1976

Design and evaluation of head unit for wheelchair control by quadriplegic patients.

A control unit was designed to allow persons who have lost hand and arm function to control the speed, steering, reversal and on-off switching of an electric wheelchair by means of backward movement and rotation of the head. When possible, shoulder movement was used to control both reversal and on-off switching. Clinical evaluation in 10 patients with quadriplegia and 2 with severe neuromuscular disease showed that the unit neither interfered with nor restrained the patients' residual body movements, permitted use of natural head movements for smooth and fast control of the wheelchair, and was well accepted by and integrated into the life of the patients.

Accident Prevention↗
PubMed2006

Engineering the quadruplex fold: nucleoside conformation determines both folding topology and molecularity in guanine quadruplexes.

Nucleic acid quadruplexes, based on the guanine quartet, can arise from one or several strands, depending on the sequence. Those consisting of a single strand are usually folded in one of two principal topologies: antiparallel, in which all or half of the guanine stretches are antiparallel to each other, or parallel, in which all guanine stretches are parallel to each other. In the latter, all guanine nucleosides possess the anti conformation about the glycosidic bond, while in the former, half possess the anti conformation, and half possess the syn conformation. While antiparallel is the more common fold, examples of biologically important, parallel quadruplexes are becoming increasingly common. Thus, it is of interest to understand the forces that determine the quadruplex fold. Here, we examine the influence of individual nucleoside conformation on the overall folding topology by selective substitution of rG for dG. We can reverse the antiparallel fold of the thrombin binding aptamer (TBA) by this approach. Additionally, this substitution converts a unimolecular quadruplex into a bimolecular one. Similar reverse substitutions in the all-RNA analogue of TBA result in a parallel to antiparallel change in topology and alter the strand configuration from bimolecular to unimolecular. On the basis of the specific substitutions made, we conclude that the strong preference of guanine ribonucleosides for the anti conformation is the driving force for the change in topology. These results demonstrate how conformational properties of guanine nucleosides govern not only the quadruplex folding topology but also impact quadruplex molecularity and provide a means to control these properties.

Guanine↗
PubMed1996

An expression system for the single-step production of recombinant human amidated calcitonin.

Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.

Amino Acid Sequence↗
PubMed2003

Activation of human telomerase reverse transcriptase expression by some new symmetrical bis-substituted derivatives of the anthraquinone.

As a part of our program aimed at exploring the biological activity of symmetrical substitution of side chains into the anthracene-9,10-dione chromophore, we have synthesized a series of 1,5-bisthioanthraquinones 2 and 1,5-bisacyloxyanthraquinones 3 that are related to the antitumor agent mitoxantrone. Since the telomerase enzyme is a novel target for potential anticancer therapy and stem cell expansion, we explore the biological effects of these compounds by evaluating their effects on telomerase activity and telomerase expression. Telomerase is required for telomere maintenance and is active in most human cancers and in germinal cells but not in most of the normal human somatic tissues. We found that most of the 1,5-disubstituted anthraquinones did not exhibit inhibitory activity at the concentration ranging from 20 to 30 microM. To facilitate the analysis of the expression of telomerase, we used cancer and normal cell lines that carry secreted alkaline phosphatase (SEAP) gene under the control of human telomerase reverse transcriptase (hTERT). The effects of these compounds on the expression of telomerease were analyzed using the cell-based reporter systems. While most of these compounds did not appear to selectively repress the expression of hTERT in cancer cells, compounds 3a, 3d, and 3i activated hTERT expression in normal cells. The effects of these three compounds on hTERT expression appear to be specific because they did not increase the expression of a CMV promoter-driven SEAP. Thus, in addition to anticancer functions, our finding raises the possibility that these compounds might also have a role in cell immortalization. The application of these anthraquinone derivatives in stem cell research and tissue engineering is also discussed.

Alkaline Phosphatase↗