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141.
Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment 总被引:2,自引:0,他引:2
Manuel Rey María Victoria González Ricardo J. Ordás Raffaela Tavazza Giorgio Ancora 《Physiologia plantarum》1996,96(4):630-636
Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment. 相似文献
142.
High levels of regeneration were obtained from young leaves excised from axillary shoots in proliferating nodal cultures of several Vitis x Muscadinia hybrids. Best results were obtained when the explants were cultured on solidified half-strength Murashige and Skoog medium supplemented with 8.9 M 6-benzyladenine and 0.05 M 1-naphthaleneacetic acid. Though variation was observed among the hybrids, the procedure used does not seem to be genotype-specific as all the hybrids and cultivars tested could regenerate. Scanning electron microscopy observations and histological studies carried out during the development of adventitious shoot organogenesis revealed that the promeristem initiation occurred in the outer cell layers near the wounded area of the petiolar stub.Abbreviations BA
6-benzyladenine
- NAA
1-naphthaleneacetic acid 相似文献
143.
Ignacio García-De La Torre Mario Salazar-Páramo Gerardo Salmón-De La Torre 《Molecular biology reports》1996,23(3-4):153-157
Mixed connective tissue disease (MCTD) was described as a distinct clinical syndrome in 1972. Since then many cases have been reported in the literature worldwide. In this study we present our experience with a group of 17 Mexican patients with this syndrome, and we analyze their clinical and serological features, as well as the causes of death in these patients. The patients are Mexican mestizos living in Guadalajara and most of them have been followed-up at Hospital General de Occidente for a period of 1–10 years. The female/male ratio was 16:1, and their age ranged from 14–55 years with a mean of 29 years. The disease duration has ranged from 1–17 years, with a mean of 6 years. Among the clinical manifestations we have found a high frequency of lymphadenopathy when compared with published series (13/17 or 76%), and the laboratory findings in our patients included a very high polyclonal increase of gammaglobulins (93%), lymphopenia (76%), direct immunofluorescence speckled nuclear epidermal deposits in skin biopsies (75%) and positive rheumatoid factor (65%). Other clinical and serological features were similar to those reported in other series of patients with MCTD. Six of the 17 patients have died (35%), and in 3 of them (17.5%) the cause of death was due to an infectious disease that suddenly presented, and apparently was not related to a concomitant high dose of steroids or malnutrition in the patients. It seems that in addition to the already well known autoimmune abnormalities that occur in MCTD, there are other features like the presence of lymphadenopathy, the high polyclonal increase of gammaglobulins, and the lymphopenia, that reflect the profound disturbance of the immune system in this syndrome, possibly contributing to the sudden appearance of a severe infectious disease in some of our patients.Abbreviations ANA
antinuclear antibody
- CIE
counterimmunoelectrophoresis
- MCTD
mixed connective tissue disease
- PHA
passive hemagglutination
- PM
polymyositis
- RF
rheumatoid factor
- SLE
systemic lupus erythematosus
- SS
systemic sclerosis (SS) 相似文献
144.
BESSESEN, DANIEL H, CONNIE L RUPP AND ROBERT H ECKEL. Dietary fat is shunted away from oxidation, toward storage in obese zucker rats. Obes Res. 1995;3:179–189. Previous measurements of lipoprotein lipase (LPL) activity in adipose tissue (ATLPL) of lean and obese Zucker rats have consistently documented increased activity in obese rats relative to lean. Since LPL is considered to be rate limiting for the delivery of triglyceride fatty acids (TGFA) to muscle and adipose tissue, these data have been used to suggest that the metabolic partitioning of TGFA favors storage over oxidation in obese rats. To document the partitioning of TGFA directly, the fate of 14C labeled oleic acid (42nmols) was fed to lean, obese, and obese Zucker rats fed a hypocaloric diet designed to chronically reduce weight 25% below that of obese controls (reduced-obese). The amount of 14C recovered in CO2 over 6 hours following ingestion was significantly less in obese rats compared to lean (0.45 ± 0.06 vs. 0.88 ± 0.09nmols, p=.0004) and less still in the reduced obese group (0.34 ± 0.06nmols p=.00003). Six hours after ingestion, the quantity of label found in adipose tissue was significantly greater in the obese rats compared to lean (14.51 ± 1.92 vs. 1.38 ± 0.29nmols p<.00001), but was intermediate in the reduced-obese group (9.23 ± 0.98nmols p=.0003). At 2.2 hours there was significantly more label in skeletal muscle of lean rats compared to either obese or reduced-obese (2.33 ± 0.24; 1.35 ± 0.04nmols p=.01; 1.41 ± 0.27nm p=.02). However, at 6 hours these differences between groups were no longer present. These findings Indicate that dietary fat is shunted away from oxidation toward storage in obese Zucker rats. Additionally it appears that there may be a relative block in the oxidation of TGFA that is taken up by skeletal muscle in obese rats. Finally the relative normalization of this partitioning defect in reduced-obese rats is at variance with what was suggested by previous measurements of tissue specific levels of LPL, and suggests an enhanced recirculation of fatty acids from adipose tissue to muscle in reduced-obese rats. This could occur through increased delivery of non-esterified fatty acids (NEFA) to muscle as a result of an increase in net lipolysis. 相似文献
145.
We have established a primary culture system for Drosophila eye imaginal discs. With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells. Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons. Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23°C. These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium (“4+5”); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS). Eye disc fragments survived in this medium for at least 20 days. Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20-hydroxyecdysone (20-HE) (1 μg/ml), whereas neurite outgrowth was seen in the absence of 20-HE. Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro. Eye discs from 5-h pupae (P+5) or older commenced ommachrome synthesis in vitro, in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies. Average neurite length was not affected by age among pupae younger than P+5; but neurite outgrowth from P+24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro. Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro. Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro. Morphogenetic changes were manifested in cultures. Viability tests with calcein AM and ethidium bromide revealed few dead cells in living cultures. © 1995 John Wiley & Sons, Inc. 相似文献
146.
利用PCR方法对单纯疱疹病毒Ⅱ型糖蛋白D(HSV-2gD)基因进行了修饰,在其5'端删去约500bp的非编码区,仅保留ATG上游7个bp。将修饰后的HSV-2gD基因插入到带有痘苗病毒天坛株TK基因区段的痘苗表达质粒pJSA1175,置于痘苗病毒P7.5k早/晚期启动子控制下。将此重组质粒用脂质体Lipofectin方法转染已受野型TK ̄+痘苗病毒天坛株感染的TK ̄-143细胞,通过同源重组机制和标志基因LacZ产物的蓝斑显色作用,以及BudR试剂对TK表型的选择压力,筛选出整合有HSV-2gD基因的重组痘苗病毒。Southem杂交表明,HSV-2gD基因已正确地插入痘苗病毒TK基因区内;间接免疫荧光检测显示,HSV-2gD蛋白已得到有效表达,且主要分布于细胞膜。重组病毒免疫家兔可产生明显的抗HSV-2gD中和抗体。用重组病毒免疫小鼠,3周后可使94%(17/18)的小鼠对抗HSV-2的致死量攻击,表明重组病毒具有明显的免疫保护作用。 相似文献
147.
Guo-Ling Nan Adelheid R. Kuehnle 《In vitro cellular & developmental biology. Plant》1995,31(3):131-136
Summary Five parameters were examined for their effect on transformation ofDendrobium tissues by microprojectile bombardment. The superpromoter in pBI426 produced at least 1.5 times as many transient transformants
as the single cauliflower mosaic virus 35S promoter in pBI121 (37 to 69% vs. 0 to 44%) with dark and frequent GUS (β-glucuronidase) staining. Tissue, genotype, and type of microparticle significantly affected transient GUS activity. Higher
expression was seen in protocormlike bodies and in hybrid UH44 compared to etiolated shoots and protocorms and to hybrids
M61 and K1329-39. Microparticles of 1.6-μm Bio-Rad gold were more effective than 1.0-μm ASI gold. Transient GUS activity did
not differ among protocormlike bodies bombarded using helium propellant pressures of 650, 900, or 1100 psi. Transgenic plants
were recovered fromDendrobium UH800 protocormlike bodies bombarded with pBI426-coated, 1.1-μm tungsten particles using an early-model gunpowder-driven
apparatus with an estimated stable transformation rate of 11.7%. One transgenic plant ofDendrobium UH44 was recovered from etiolated shoot explants bombarded with pBI121-coated, 1.1-μm tungsten particles using the Dupont
PDS-1000 with a stable transformation rate of 0.17%. Positive selection results showed 100 to 200 mg·liter−1 kanamycin to be appropriate for regeneration of transgenic plants from protocormlike bodies, protocorms, and etiolated shoot
explants over a 3- to 9.5-mo. period. 相似文献
148.
Alvaro N. A. Monteiro Radovan Borojevic 《In vitro cellular & developmental biology. Animal》1995,31(2):149-155
Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics
of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of
different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of
fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation
of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native
complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic
livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion
of connective tissue cells during the development of liver fibrosis. 相似文献
149.
Ralph Rapley 《Molecular biotechnology》1995,3(2):139-154
The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the
in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However,
many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application
to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular
techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies
themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and
modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine
antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression
systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering
and in the successful development of new diagnostic and therapeutic antibody-based reagents. 相似文献
150.
Ramana M. Gosukonda Ananta Porobodessai Essie Blay Channapatna S. Prakash Curt M. Peterson 《In vitro cellular & developmental biology. Plant》1995,31(2):65-71
Adventitious shoots of sweetpotato (Ipomoea batatas L. Lam.) were produced in vitro using a two-stage culture method. Petiole explants were incubated on Murashige and Skoog
(MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (0.2 mg·liter−1) for 3 d, and transferred to MS medium with thidiazuron (0 to 0.4 mg·liter−1). Shoot regeneration was observed in most explants (78.2%) of genotype PI 318846-3 within 28 days when cultured on thidiazuron
at 0.2 mg·liter−1. Histological studies of cultured petiole explants showed meristematic activity within cells of vascular bundles and throughout
the ground tissue. Explants isolated from apical leaves exhibited higher shoot regeneration frequency than those isolated
from the basal portion of the shoot. Leaf lamina explants exhibited lower frequency of regeneration than petiole explants.
In contrast to thidiazuron, the use of zeatin riboside, and kinetin resulted in a lower frequency of shoot regeneration although
more sweetpotato genotypes could be regenerated using either of these two cytokinins. The sweetpotato plants regenerated using
thidiazuron grew vigorously and rooted easily when transferred to the greenhouse. 相似文献