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PubMed2004

Characterization of apoM in normal and genetically modified mice.

A novel human apolipoprotein [apolipoprotein M (apoM)] was recently described and demonstrated to be a lipocalin. We have now examined apoM in wild-type mice and mice with genetically altered lipoprotein metabolism. Liver and kidney showed high mRNA expression, whereas spleen, heart, brain, and testis demonstrated low expression. ApoM gene expression was initiated on embryonic day 10. Western blot analysis of plasma suggested that mouse apoM, like its human counterpart, is secreted with a retained signal peptide, but unlike human apoM it is not glycosylated. Gel filtration of plasma showed apoM to be associated with HDL-sized particles in wild-type and apoA-I-deficient mice and with HDL- and LDL-sized particles in LDL receptor-deficient mice, whereas apoM was mainly found in VLDL-sized particles in high-fat, high-cholesterol-fed apoE-deficient mice. The plasma concentration of apoM was similar in wild-type, LDL receptor-deficient, and apoE-deficient mice but was reduced to 33% in apoA-I-deficient compared with wild-type mice (P = 0.007). These data suggest that apoM mainly associates with HDL in normal mice but also with the pathologically increased lipoprotein fraction in genetically modified mice. The substantially decreased apoM levels in apoA-I-deficient mice suggest a connection between apoM and apoA-I metabolism.

Animals↗
PubMed2001

Changes in tissue cellularity are associated with growth enhancement in genetically modified arctic char (Salvelinus alpinus L.) carrying recombinant growth hormone gene.

Biochemical and histological analyses were used to study the number and size of cells (cellularity) in tissues of fast-growing, genetically modified Arctic char (Salvelinus alpinus L.), overexpressing sockeye salmon (Oncorhynchus nerka) growth hormone gene (OnGH1). DNA contents of muscle, heart, and liver were compared in transformed, sibling (age control) and 1 year older (size control) char. Total white muscle cross-sectional area, white muscle fiber number, and total nuclei number within the muscle tissue were determined from one complete half-section of each fish. The analyzed tissues responded differently to growth hormone overproduction. In muscle tissue of OnGH1-transformed char, the enhanced growth was clearly associated with proliferation of muscle cells (hyperplasia), whereas in heart tissue both cell proliferation and increase in cell size (hypertrophy) were enhanced. The relative DNA concentration in the liver of transformed char was significantly greater than that of control fish, suggesting reduction in size of hepatic cells.

Journal Article↗
PubMed1995

Genetically modified fibroblasts producing NGF protect hippocampal neurons after ischemia in the rat.

The neuroprotective effect of nerve growth factor (NGF) on the pyramidal cells in the vulnerable CA1-CA2 sectors of the hippocampus was investigated in a rat model of transient forebrain ischemia. A genetically modified fibroblast line that secretes high levels of NGF was implanted 7 days before induction of ischemia between the hippocampal CA1-CA2 subfields in the right hemisphere. Rats were then subjected to 10 min of cerebral ischemia in a four vessel occlusion model. Morphological changes in the CA1 and CA2 subfields were evaluated 7 days after ischemia. Animals in the NGF-protected group had significantly higher numbers of normal appearing neurons in the right CA1 and CA2 regions, compared with their non-implanted left hemispheres, to non-implanted animals or to animals implanted with non-modified cells. The data confirmed that NGF can protect CA1-CA2 hippocampal neurons from ischemic damage by implantation of genetically engineered cells producing NGF.

Animals↗
PubMed2006

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.

Arsenic↗
PubMed2000

Phosphorus bioavailability and digestibility of normal and genetically modified low-phytate corn for pigs.

We conducted two studies to determine the bioavailability and apparent digestibility of P in a low-phytate corn hybrid (.28% total P, .10% phytate P) genetically modified to be homozygous for the 1pa1-1 allele and a nearly isogenic corn hybrid (normal) (.25% total P, .20% phytate P). Additionally, we conducted an in vitro assay involving a peptic and pancreatin digestion to estimate P availability. The first study used 50 individually penned pigs (initial body weight 9 kg) and 10 treatments in a randomized complete block design. A cornstarch-soybean meal basal diet (.6% Ca, .2% P) was used. Treatments consisted of the basal diet and the basal diet plus .05, .10, or .15% P from monosodium phosphate (MSP), low-phytate corn, or normal corn. After a 35-d feeding period, pigs were killed to collect the fourth metacarpal for measurements of ash and breaking load. Breaking load was regressed on added P intake, and the bioavailability of P was determined by the slope ratio method. The bioavailabilities of P (relative to MSP) for low-phytate and normal corn were 62 and 9%, respectively. These were similar to the determined in vitro values of 57 and 11% for low-phytate and normal corn, respectively. In the second study, 20 pigs (initial BW 20 kg) were used in a randomized complete block design with a 2 x 2 factorial arrangement of treatments. Two corn lines (low-phytate and normal) and two levels of supplemental P (0 and .2%) from dicalcium phosphate were used. Diets with no added P were formulated to contain .9% lysine, .6% Ca, and .34% P. Apparent nutrient digestibilities were calculated from total collection of urine and feces for 5 d. There were no differences among treatments for energy and nitrogen digestibility. Pigs fed low-phytate corn with no added P had increased digestibility and retention of P and reduced total P excretion (P < .05). We conclude that low-phytate corn contains at least five times as much available P as normal corn. The use oflow-phytate corn greatly reduced the amount of P excreted by the pig and increased the N:P ratio in the manure.

Animal Feed↗
PubMedDate not supplied

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo.

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 x 10(3) to 1.5 x 10(5) cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 micrograms/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

Animals↗
PubMed2003

Tumor necrosis factor-alpha production-regulating activity of phthalimide derivatives in genetically modified murine melanoma cells B78H1.

The effect of imides, monothioimides, trimellitimides, as well as 5'-deoxy-5'-phthaloylamino-derivatives of azidothymidine on tumor necrosis factor-alpha (TNF-alpha) production by genetically modified murine B78H1 melanoma cells transduced with the gene for human TNF-alpha (B78/TNF) was investigated. It was found that N-(adamant-1-yl)monothiophthalimide (1e) and N-(adamant-2-yl)-monothiophthalimide (1f) showed over 200% enhancing of TNF-alpha production while some of imides were inhibitors.

Animals↗
PubMed2001

Environmental risks of chemicals and genetically modified organisms: a comparison. Part II: Sustainability and precaution in risk assessment and risk management.

The principles of precaution and sustainability require more consideration in the assessment of environmental risks posed by chemicals and genetically modified organisms. Instead of applying risk reduction measures when there are serious indications for damage, full scientific certainty is often waited for before taking action. The precautionary principle particularly should be applied in those cases in which the extent and probability of damage are uncertain, e.g. in the case of persistent chemicals which are additionally bioaccumulative or highly mobile. Based on these principles, environmental action targets for risks associated with GMOs and chemicals can be developed. Risk management not only includes statutory measures but also instruments designed to influence behaviour indirectly are important to achieve the goals. Particularly for risks of GMOs which provoke fear, risk communication is important. Some rules to which attention should be paid in communication with the public are presented.

Animals↗
PubMed2005

The splicing machinery is a genetic modifier of disease severity.

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.

Alternative Splicing↗
PubMed2001

Compatibility of a wild type and its genetically modified Sinorhizobium strain with two mycorrhizal fungi on Medicago species as affected by drought stress.

The effect of double inoculation with two strains of Sinorhizobium meliloti [the wild type (WT) strain GR4 and its genetically modified (GM) derivative GR4(pCK3)], and two species of arbuscular mycorrhizal (AM) fungi (Glomus deserticola and Glomus intraradices) was examined in a microcosm system on three species of Medicago (M. nolana, M. rigidula, M. rotata). Two water regimes (80 and 100% water holding capacity, WHC) were assayed. The efficiency of each AM fungus increasing plant growth, nutrient content, nodulation and water-stress tolerance was related to the Sinorhizobium strains and Medicago species. This indicates selective and specific compatibilities between microsymbionts and the common host plant. Differential effects of the mycorrhizal isolates were not associated with their colonizing ability. Nodulation and mycorrhizal dependency (MD) changed in each plant genotype in accordance with the Sinorhizobium strain and AM fungi involved. Generally, Medicago sp. MD decreased under water-stress conditions even when these conditions did not affect AM colonization (%). Proline accumulation in non-mycorrhizal plant leaves was increased by water stress, except in M. rotata plants. Differences in proline accumulation in AM-colonized plants suggest that both the AM fungus and the Sinorhizobium strain were able to induce different degrees of osmotic adjustment. Mycorrhizal plants nodulated by the WT strain accumulated more proline in M. rigidula and M. rotata under water stress than non-mycorrhizal plants. Conversely, mycorrhizal plants nodulated by the GM strain accumulated less proline in response to both AM colonization and drought. These results indicated changes in the synthesis of this nitrogenous osmoregulator product associated with microbial inoculation and drought tolerance. Mycorrhizal plants nodulated by the GM Sinorhizobium strain seem to suffer less from the detrimental effect of water stress, since under water limitation relative plant growth, percentage of AM colonization, root dry weight and the highest R/S ratio remained the same. The fact that GM nodulated plants are better adapted to drought stress could be of practical interest and the management of GM microorganism inoculation may be crucial for biotechnological approaches to improving crop yield in dry environments.

Journal Article↗
PubMed2000

Transplantation of normal and genetically modified adrenocortical cells.

Cell transplantation techniques have been applied to the study of the biology of the adrenal cortex and to adrenocortical cell proliferation, differentiation, and senescence. Primary bovine adrenocortical cells, primary human adrenocortical cells and genetically modified bovine adrenocortical cells have been transplanted. Successful methods include transplantation of cells beneath the kidney capsule and several subcutaneous cell transplantation procedures. In successful transplants the cells form a functional vascularized tissue structure that allows the host animals to survive adrenalectomy. We show here that subcutaneous cell transplantation does not depend on embedding cells in collagen gel before introduction into the host animal. Subcutaneous transplants secrete both cortisol and aldosterone. However, the variability of plasma aldosterone levels indicates that the factors that determine glomerulosa-type and fasciculata-type cell function in transplant tissues are not well understood.

3T3 Cells↗
PubMedDate not supplied

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules.

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.

DNA, Plant↗
PubMed1999

Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Acetylcholine↗
PubMed2000

Genetically modified tumour vaccines (GMTV) in melanoma clinical trials.

Since melanoma is a model immunogenic malignancy incurable in the disseminated phase of its natural course different immunotherapeutic approaches are tested in clinical trials. A number of tumour vaccines genetically modified (GMTV), with various immunostimulatory factors, are tested in phase I/II clinical trials. These factors include cytokines, tumour antigens (TA), costimulatory molecules or HLA antigens. We have designed a novel, mixed auto/allogeneic cellular melanoma vaccine modified with the IL-6 and the sIL-6R genes. Preclinical studies in a mouse model demonstrated that the IL-6/sIL-6R based vaccine is able to elicit efficient anti-tumour responses, mediated by CD8+ and NK cells, which resulted in inhibition of the tumour growth, metastases formation and prolonged survival of the animals treated. Irradiation of vaccine cells does not only lead to their sterilisation but also causes increased secretion of exogenous IL-6 and sIL-6R. Since January 1996 we have vaccinated more than one hundred metastatic melanoma patients. Promising clinical results (22% CR+PR, 32% SD) and the evidence of immune responses in the vaccinated patients have prompted us to design a phase III clinical trial which is to be open in 2000.

Animals↗
PubMed1996

Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures.

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.

Animals↗
PubMed2004

Genetically modified mice as a tool to study inflammatory skin diseases.

Although mice have a long tradition as models for human skin diseases, they have recently received increasingly more attention. This is because of the rapid advancement of genetic engineering methods which made it possible to create mice with precisely defined genetic changes. Many of these mice develop impressive and sometimes unexpected, puzzling phenotypes. Their interpretation is a major challenge to the basic researcher and can often be ameliorated by the input of an experienced dermatologist. Together with recent examples of genetically modified mouse models of inflammatory skin diseases we give a short overview of the methods used to generate such mice, describe possible ways to analyse them, and discuss problems that arise in the interpretation of the findings.

Animals↗