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PubMed2004

Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori.

In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother. On the other hand, it is essential to obtain continuous generations (polyvoltinism) after microinjection, in order to screen positive transgenic insects and study genetics and insertion of the transgene. Thereafter, it is more convenient to store these populations, as diapause eggs before their use in biotechnology application. We obtained such polyvoltine parthenoclones, first by selection for a parthenogenetic character in polyvoltine races, and second, by selection for a polyvoltine character in a parthenogenetic, but diapausing clone of B. mori. As diapause was directly under the control of diapause hormone (DH), we also tested direct injection of DH in female pupae of polyvoltine strains, as well as anti-DH antibody treatment to eliminate diapause in univoltine strains. We discussed the advantages and limitations of these methods and proved the feasibility in obtaining polyvoltine parthenoclones and determining the voltinism in B. mori. These methods would permit us to improve the management of populations used in transgenesis technology.

Animal Husbandry↗

PubMedDate not supplied

Regulation of myosin heavy chain and actin isogenes expression during cardiac growth.

The cardiac ventricular myosin heavy chain phenotype is developmentally and hormonally regulated, but less is known concerning the actin phenotype. In this study, the levels of accumulation of alpha-skeletal and alpha-cardiac actin mRNAs were investigated in rat and human ventricles by primer extension assays. In rat, the two iso-mRNAs are present in approximately equal amounts from birth until 15 days of age and the cardiac form is predominant in adult and senescent hearts. Hypothyroid development has no effect, at least during the first two weeks of age. In man, the two isoactins are co-expressed to similar ratios in one control heart and in one failing heart. It therefore appears that myosin heavy chain and actin multigene families are both expressed in a species specific fashion but are independently regulated within a species. Preliminary results from nuclear run-on assays are presented that indicate differences in the level of transcription of the alpha-actin and beta-myosin heavy chain isogenes in the rat heart.

Actins↗

PubMed2004

Identification of candidate genes for in vitro androgenesis induction in maize.

Extensive studies have been conducted to understand the genetic control of in vitro androgenesis, but little is know about the genes and the mechanisms involved in the switch that allows an immature pollen grain to develop as an embryo. We have developed two maize isogenic lines with high androgenetic aptitude, named AH5-44 and AH5-49, through backcross and selection from a high-responsive DH229 line on the non-responding A188 line genetic background. The genomic structure of these two lines was precisely described with microsatellite markers. Five regions retained from the parent DH229 highly responsive to androgenesis were localised in both AH5-44 and AH5-49. Sequences expressed on microspores extracted from the four lines were amplified using a cDNA-AFLP protocol. For each line, eight culture conditions were compared: microspores extracted after tassel recovery, after 7 or 14 days in cold room and after 1-4 days of in vitro culture. This genetic and developmental screening allowed us to identify four sequences, including a new HSP70-like candidate gene. Possible implication of the identified sequences in androgenesis response is discussed.

Base Sequence↗

PubMed1981

Effects of maternal anti H-Y antibodies on fetal gonadal development.

C57BL/6 inbred female mice preimmunized by serial injections of isogeneic male spleen cells were mated and the effects of the circulating anti H-Y antibodies on the fetal gonads and on their fertility were studied. Female mice receiving injections of saline formed one control group and those given female spleen cells a second control group. The experiment was carried out three times and since observations were similar and reproducible the data of all three sets were pooled. There was no significant change in the birth weights of newborns, total number of conceptions or sex ratio in the study group compared to either of the controls. There was no difference in the mean live birth rate between the study group and both the controls. Although the incidence of stillbirths in the study group was higher than that in both controls the difference was not statistically significant. In the study group there were no histopathological changes in the gonads from female pups compared to ovaries from the controls. In contrast, the gonads of male pups showed mild to severe structural disorganization in the study group, but not in the female spleen cell injected group. In spite of the morphological changes in the testes, the newborn male pups had a normal anogenital distance and testicular descent suggesting that Leydig cell function was normal.

Animals↗

PubMedDate not supplied

Contractile activity of circular smooth muscle in rats one year after small bowel transplantation: differing adaptive response of the jejunum and ileum to denervation.

The aim of the present study was to determine the long-term effects of isogeneic small bowel transplantation (SBT) on jejunal and ileal circular smooth muscle contractile activity in the rat. Transmural strips of circular muscle were prepared from proximal jejunum and distal ileum of 1-year-old control rats and rats 1 year after SBT (SBT-1Y) to measure isometric force. Spontaneous contractile activity and the dose-responses to bethanechol and norepinephrine were studied. Electrical field stimulation (EFS) at varying frequencies (1 to 20 Hz) was evaluated under adrenergic and cholinergic blockade to investigate inhibitory nerves. Spontaneous activity both in the jejunum and ileum in SBT-1Y rats was not different compared to control rats. Sensitivity to bethanechol did not differ between control and SBT-1Y rats in the jejunum or ileum. Sensitivity to norepinephrine, however, was significantly increased after SBT in the ileum but not in the jejunum. During EFS, inhibition was seen at low frequencies, and contractions were induced at high frequencies in all groups. The degree of inhibition did not differ between control and SBT-1Y rats in the jejunum; however, it tended to be increased in the ileum after SBT. The long-term adaptive response of smooth muscle to the extrinsic denervation accompanying SBT differs between the jejunum and the ileum.

Adaptation, Physiological↗

PubMed1999

The influence of cortical perforations and of space filling with peripheral blood on the kinetics of guided bone generation. A comparative histometric study in the rat.

The aim of the present study was to evaluate the influence of cortical perforations and of peripheral blood addition in guided bone generation beyond the skeletal envelope in rats. A total of 30 isogenic adult rats were divided into 3 equal groups. In each rat, two hollow parallelipipedic titanium chambers were placed bilaterally on the calvaria after a periosteal skin flap was raised. While on the right sides (controls) the osseous surface was left intact and the chambers were empty, the cortical bone under the left-side chambers (test sites) was perforated with nine 0.8 mm-diameter holes (group I), or left intact but with the chambers filled with a clot of peripheral blood (group II). In group III, both procedures were combined in the test sites. The healing was assessed at 4, 8 and 16 weeks after surgery by histologic and computer-assisted histometric analysis. The results demonstrated a substantial augmentation of on average 141% (SD 18) of the skull's thickness after 16 weeks in the controls, indicating that a predictable bone formation can be achieved beneath completely occlusive barriers over a non-injured cortical layer. In all test groups, a significantly larger bone augmentation was observed after 16 weeks compared to the control sites 172.8% (SD 41.7) in group I (P < 0.05), 172.0% (SD 18.4) in group II (P < 0.05) and 221.5% (SD 42.3) in group III (P < 0.001), demonstrating that stimulating blood supply and bone forming cells access by cortical perforations and/or blood clot addition enhances de novo bone formation in this experimental model.

Animals↗

PubMed1994

Signaling among neighboring plants and the development of size inequalities in plant populations.

Transgenic tobacco plants that express an oat phytochrome gene (phyA) under control of the cauliflower mosaic virus (CaMV) 35S promoter and display altered photophysiology were used to test the role of light as a vehicle of information in dominance relationships between neighboring plants. Compared with the isogenic wild type, phyA-overexpressing plants showed dramatically reduced morphological responsivity to changes in the red/far red ratio of the incident light and to the proximity of neighboring plants in spacing experiments. In transgenic canopies an increase in stand density caused the small plants of the population to be rapidly suppressed by their neighbors. In wild-type canopies, plants responded to increased density with large morphological changes, and there appeared to be an inverse relationship between the magnitude of this morphological response and the ranking of the individual plant in the population size hierarchy. In these wild-type populations, size inequality increased only moderately with density within the time frame of the experiments. Our results suggest that, in crowded stands, the ability of individual plants to acquire information about their light environment via phytochrome plays a central role in driving architectural changes that, at the population level, delay the development of size differences between neighbors.

Avena↗

PubMed2002

Inhibition of intracellular macromolecular synthesis in Staphylococcus aureus by thrombin-induced platelet microbicidal proteins.

Thrombin-induced platelet microbicidal proteins (tPMP-1 and tPMP-2) are believed to initiate their staphylocidal effects via cytoplasmic membrane perturbation. The aim of the present study was to investigate the role of subsequent inhibition of macromolecular synthesis in the staphylocidal mechanisms of tPMP-1 and tPMP-2 in an isogenic tPMP-susceptible and -resistant strain pair (ISP479C and ISP479R, respectively). In ISP479C, tPMP-1 and tPMP-2 (2 microg/mL) exerted significant bactericidal effects and significantly reduced DNA and RNA synthesis (P <.05 vs. control). In contrast, tPMP-1 and tPMP-2 exerted reduced staphylocidal effects and significantly reduced inhibition of DNA and RNA synthesis against ISP479R, as compared with ISP479C (P <.05). However, tPMP-1 and tPMP-2 (2 microg/mL) caused equivalent degrees of inhibition of protein synthesis in both ISP479C and ISP479R. Collectively, these observations are consistent with the hypothesis that inhibition of specific macromolecular synthesis pathways is integral to the overall staphylocidal mechanism(s) of tPMPs.

Animals↗

PubMed2002

Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system.

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

Animals↗

PubMed2002

Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying.

The hypothesis that ABA produced by roots in drying soil is responsible for stomatal closure was tested with grafted plants constructed from the ABA-deficient tomato mutants, sitiens and flacca and their near-isogenic wild-type parent. Three types of experiments were conducted. In the first type, reciprocal grafts were made between the wild type and sitiens or flacca. Stomatal conductance accorded with the genotype of the shoot, not the root. Stomates closed in all of the grafted plants in response to soil drying, regardless of the root genotype, i.e. regardless of the ability of the roots to produce ABA. In the second type of experiment, wild-type shoots were grafted onto a split-root system consisting of one wild-type root grafted to one mutant (flacca or sitiens) root. Water was withheld from one root system, while the other was watered well so that the shoots did not experience any decline in water potential or loss of turgor. Stomates closed to a similar extent when water was withheld from the mutant roots or the wild-type roots. In the third type of experiment, grafted plants with wild-type shoots and either wild-type or sitiens roots were established in pots that could be placed inside a pressure chamber, and the pressure increased as the soil dried so that the shoots remained fully turgid throughout. Stomates closed as the soil dried, regardless of whether the roots were wild type or sitiens. These experiments demonstrate that stomatal closure in response to soil drying can occur in the absence of leaf water deficit, and does not require ABA production by roots. A chemical signal from roots leading to a change in apoplastic ABA levels in leaves may be responsible for the stomatal closure.

Abscisic Acid↗

PubMed2005

The discovery,genetic analysis and gene mapping of earliness rice (Oryza sativa L.) D64B.

D64B, an achromatic early mutation, was discovered from indica rice preserver line D63B. D64B were crossed with inhomogeneous sterile lines, preserver lines and restorer lines, most of their F1 shared the same heading dates as D64B. The results indicated that D64B possessed the characteristic of dominant earliness. To analyze the earliness trait, positive and negative crosses F2 and BC1 from the cross of D64B and Shuhui 527 were cultivated. Their heading dates showed double apices distributing with same apex values. According to apex vale value -103 d, plants of the F2 and BC1 were separated into early plants and late plants, which were tested with Chi2 value. The results suggested that the segregation ratio of number of early to late plant fitted to 3:1 and 1:1 for positive and negative cross F2 and BC1, respectively, and that the earliness of D64B was controlled by a single dominant major gene. To map the gene, the polymorphisms between D64B and Shuhui 527 was analyzed with reported 356 SSR primers and 59 primers showed polymorphisms. The earliness-lateness near isogenic pools, early plants and late plants from F2 of Shuhui 527 x D64B were further amplified with the 59 primers. The results indicated that there were polymorphisms on both RM279 and RM71. Their results being analyzed with MAPMAKER/EXP3.0 software, the dominant major gene was located on the top arm of rice chromosome 2 and between the two SSR markers, RM279 and RM71, with genetic distance of 12.6 cM and 13.3 cM, respectively. According to reported data, the gene was first discovered and tentatively named as Ef-3(t). In breeding practice, sterile line D64A had been bred with D64B.

Chromosome Mapping↗

PubMedDate not supplied

Effects of iso and xeno fetal liver fragments transplantation on acute and chronic liver failure in rats.

Isogeneic (rat) and xenogeneic (swine) fetal liver fragments (FLF) transplantation into the omentum was performed for D-galactosamine (D-Gal)-induced acute and carbon tetrachloride (CCl4)-induced chronic hepatic failure in rats. The recipients that had iso or xeno FLF showed higher survival rates than the nontransplanted controls on a lethal dose (2.6 g/kg body weight) of D-Gal (survival rates: Iso 70%, Xeno 80%, and control 9.1%). On a sublethal dose (1.0 or 1.2 g/kg) of D-Gal, iso, or xeno FLF caused marked improvement of the values of GPT, GOT, and total bilirubin (T.Bil); at 72 h after D-Gal injection they went significantly lower than those of controls (Iso vs. control; p < 0.01, Xeno vs. control; p < 0.05). Histological examination of the livers revealed severe damage in controls, however, only a slight damage was found in iso or xeno FLF transplanted rats. Iso grafts were fairly well preserved in the omentum at 72 h posttransplants, however, xeno graft had almost changed into a necrotic tissue. CCl4 was administered subcutaneously for 14 wk to induce chronic hepatic failure and then iso FLF were transplanted 3 days after the last CCl4 injection. Iso FLF transplanted rats showed higher improvement of GPT and GOT values at 12 days posttransplants compared with controls (GPT p < 0.01, GOT p < 0.05), although histological improvement was not so remarkable in both group. Iso grafts formed nodules with many hepatocytes in the omentum 12 days posttransplant. The results indicate that iso or xeno FLF transplantation could be an alternative approach for incurable liver insufficiencies.

Animals↗

PubMed2005

Central autonomic control of the bone marrow: multisynaptic tract tracing by recombinant pseudorabies virus.

Bone marrow is the primary place of hematopoiesis, where the development, survival and release of multipotent stem cells, progenitors, precursors and mature cells are under continuous humoral and neural control. Dense network of nerve fibers, containing various neurotransmitters is found in the bone marrow, however, the central neuronal circuit that regulates the activities of the bone marrow through these fibers remained unexplored. Transsynaptically connected neurons were mapped by virus-based transneuronal tracing technique using two isogenic, genetically engineered pseudorabies viruses, Bartha-DupGreen and Ba-DupLac expressing green fluorescent protein and beta-galactosidase, respectively. Bartha-DupGreen was injected into the femoral bone marrow of male rats and the progression of infection was followed 4-7 days post-inoculation. Virus-labeled cells were revealed in ganglia of the paravertebral chain and in the intermediolateral cell column of the lower thoracic spinal cord. Neurons were retrogradely labeled in the C1, A5, A7 catecholaminergic cell groups and several other nuclei of the ventrolateral and ventromedial medulla, the periaqueductal gray matter, the paraventricular and other hypothalamic nuclei, and in the insular and piriform cortex. Nerve transections and double-virus tracing from the bone marrow and the surrounding muscles were used to confirm the specific spreading of the virus. These results provide anatomical evidence for the CNS control of the bone marrow and identify putative brain areas, which are involved in autonomic regulation of the hematopoiesis, the release of progenitor cells, the blood supply and the immune cell function in the bone marrow.

Animals↗

PubMed1999

Contextual effects of transforming growth factor beta on the tumorigenicity of human colon carcinoma cells.

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.

Animals↗

PubMed2001

Initial inhibition of tissue factor signalling reduces chronic vascular changes in isogenic rat aortic transplants.

Vascular changes are considered the major histopathological indicator of chronic allograft dysfunction. These changes are characterized by intimal thickening caused by accumulation of primarily smooth muscle cells. Contributing factors may be of both immunological and nonimmunological origin. Cold ischemia has been shown to trigger intimal proliferation in the absence of alloantigen in an isogenic rat aortic transplant model. We have used this model to investigate the impact of inhibition of tissue factor (TF) signalling on the progression of intimal thickening. Group 1 was treated with recombinant FVIIa inhibited in its active site (rFVIIai), and group 2 served as untreated controls. At 8 weeks the intimal area was measured with image analysis. Medial areas and the proportion of medial necrosis were determined. Animals treated with rFVIIai showed significantly less intimal thickening compared with controls: median 0.147 vs. 0.256 mm2, respectively (p = 0.008). A positive correlation between intimal hyperplasia and medial necrosis (r(s) = 0.79, p = 0.01), as well as adventitial inflammation (r(s) = 0.83, p = 0.009), was found. TF mRNA was not detected in the neointima at 8 weeks, as determined by in situ hybridization. We conclude that active site inhibited FVIIa (rFVIIai) given prior to and directly after implantation of aortic transplants significantly reduces intimal hyperplasia caused by nonimmunological factors in this model.

Animals↗

PubMed1980

Properties of Bacillus subtilis 168 derivatives freed of their natural prophages.

An isogenic set of Bacillus subtilis 168 strains which are non-inducible for prophage PBSX and are cured of prophage SP beta has been constructed. By utilizing these strains, prophage SP beta has been shown to control the inducible DNA modification system which exists in this bacterium. However, neither the PBSX nor the SP beta prophages alter the ability of the bacterium to undergo genetic recombination, to repair damaged DNA or to sporulate. Prophageless B. subtilis would be a useful host for the phi 3T cloning vector, because of the absence of vector--prophage interactions.

Bacillus subtilis↗

PubMed1997

Bone graft incorporation after cortical perforations of the host bed.

A fundamental issue in onlay bone graft persistence is the unpredictable extent of incorporation and volumetric maintenance of the graft. The purpose of this study was to evaluate the effects on integration of onlays, with either their cancellous or cortical portion facing toward the host bed, positioned over cortical perforations at the recipient site. Tibial or femoral unicortical bone grafts were harvested from isogeneic donors and positioned subperiostally on each tibia of 22 adult Lewis rats. On the experimental side, the recipient outer cortical bone surface received multiple perforations, 0.25 mm in diameter. The contralateral side served as a control (no cortical perforations). The findings were assessed after 4 and 20 weeks using routine histologic and immunohistochemistry techniques. Cortical perforations induced a migration of the recipient bone marrow into the graft as well as a reduced size diminution. More cortical bone remodeling and marginal lamellar bone apposition were observed after orientating the cortical portion of the graft toward the recipient site. These observations may be useful clinically to improve long-term success after autogeneic bone grafting.

Animals↗

PubMed2000

Influence of Helicobacter pylori on reactive oxygen-induced gastric epithelial cell injury.

Risk factors for gastric cancer are receiving renewed attention in light of the recent positive association of Helicobacter pylori infection with gastric cancer. The effect of H.pylori on the balance between oxidants and antioxidants in the stomach is not well known. In this study, we investigated if exposure of gastric cells to H. pylori increases oxidant-associated gastric epithelial cell injury. A human gastric epithelial cell line (AGS) was grown on 96-well clusters, then exposed overnight to either live H.pylori (four cagA(+) and four cagA(-)) or broth culture supernatant from an isogenic H.pylori cagA(+) strain with and without vacA activity. Incubation of AGS cells with cagA(+) and cagA(-) H.pylori strains before exposure to reactive oxygen species (ROS) reduced cell viability on average to 73.7% and 39.5% of controls, respectively. The percent viability of cells exposed to ROS after incubation with control broth, vacA(-) broth and vacA(+) broth was 97.7%, 70.5% and 63.5%, respectively. Experiments were then performed to evaluate the effects of H.pylori exposure on the activities of ROS-scavenging enzymes [catalase, glutathione peroxidase and superoxide dismutase (SOD)] and formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) adducts in AGS cells. Overnight exposure to cagA(-) strains reduced catalase activity by 42%; in contrast, exposure to cagA(+) H.pylori strains increased catalase activity by 51%. Glutathione peroxidase activity increased with exposure to both cagA(-) and cagA(+) strains by 95% and 240%, respectively. Total SOD activity increased 156% after exposure to cagA(+) strains and was marginally increased (52%) with exposure to cagA(-) strains. CuZn-SOD protein levels, assayed by enzyme-linked immunosorbent assay, were not significantly altered by exposure to H.pylori strains; however, Mn-SOD concentrations were significantly increased (P: < 0.02) after exposure to both cagA(-) and cagA(+) H.pylori strains. Exposure of AGS cells to cagA(+) and cagA(-) H.pylori was associated with, on average, 44.5 and 99.0 8-OH-dG/10(6) dG, respectively. The increase in catalase, glutathione peroxidase and SOD activity is associated with fewer 8-OH-dG DNA adducts and reduced susceptibility of AGS cells to lethal injury from ROS after exposure to cagA(+) H.pylori strains when compared with exposure to cagA(-) H.pylori strains. Alteration in the activity of ROS-scavenging enzymes by the presence of H. pylori may in part be responsible for the increased risk of gastric cancer in persons infected with H.pylori.

Antigens, Bacterial↗