Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

Summary Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + Keen sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) Valencia sweet orange (C. sinensis (L.) Osbeck) + Femminello lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.Abbreviations cp chloroplast - GOT glutamateoxaloacetate transaminase - IDH isocitrate dehydrogenase - mt mitochondria - PEG polyethylene glycol - PGI phosphoglucose isomerase - PGM phosphoglucomutase - RFLP restriction fragment length polymorphism Florida Agricultural Experiment Station Journal Series No. R-04631.     

A mini organ culture as a model for studying the gallbladder epithelium of mouse

Summary— A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

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.     

Adventitious bud formation and shoot development from in vitro leaves of Vitis x Muscadinia hybrids

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     

Mixed connective tissue disease. A clinico-serological study of 17 cases

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)     

Dietary Fat Is Shunted Away from Oxidation,Toward Storage in Obese Zucker Rats

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.     

Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster

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.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

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.     

Effects of T3 and rearing temperature on growth and skeletal myosin heavy chain isoform transition during early development in the salmonid Salvelinus alpinus (L.)

Rearing of 1-year-old Arctic charr (Salvelinus alpinus) at 12°C, as well as the administration of 50 or 75 mgT₃/kg feed, accelerated the neonatal to adult fast myosin heavy chain transition, but the effect of temperature was more dramatic than the effect of T₃ administration. The endogenous plasma levels of T₃ in charrs reared at 12°C were higher than those of analogous groups reared at natural temperature, which in the period under study was between 0.5 and 12°C. As in other species, T₃ seemed to play a role in the regulation of the neonatal to adult fast myosin isoform transition by down-regulating the levels of the neonatal and increasing the levels of an adult fast myosin heavy chain. Temperature seemed to accelerate this transition at least, but not only, by inducing an increase in the endogenous levels of T₃ in the Arctic charr.