The tomato dark green mutation is a novel allele of the tomato homolog of the DEETIOLATED1 gene.

A comprehensive, multi-generation, allele test, carried out in this study, suggests that the tomato mutations dark-green (dg) and high pigment 2(j) (hp-2(j)) are allelic. The hp-2(j) mutant is caused by a mutation in the tomato homolog of the DEETIOLATED1 (DET1) gene, involved in the signal transduction cascade of light perception and morphogenesis. This suggestion is in agreement with the exaggerated photomorphogenic de-etiolation response of homozygous dg mutants grown under modulated light conditions. Sequence analysis of the DET1 gene was carried out in dg mutants representing two different lines, and revealed a single A-to-T base transversion in the second exon of the DET1 gene in comparison with the normal wild-type sequence. This transversion results in a conserved Asparagine(34)-to-Isoleucine(34) amino-acid substitution, and eliminates a recognition site for the AclI restriction endonuclease, present in the wild-type and in the other currently known tomato mutants at the DET1 locus. This polymorphism was used to develop a PCR-based DNA marker, which enables an early genotypic selection for breeding lycopene-rich tomatoes. Using this marker and sequence analysis we demonstrate that an identical base transversion also exists in dg mutants of the cultivar Manapal, in which the natural dg mutation was originally discovered. A linkage analysis, carried out in a F(2) population, shows a very strong linkage association between the DET1 locus of dg mutant plants and the photomorphogenic response of the seedlings, measured as hypocotyl length (12 < LOD Score < 13, R(2) = 51.1%). The results presented in this study strongly support the hypothesis that the tomato dg mutation is a novel allele of the tomato homolog of the DET1 gene.

FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco.

Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants. Here we characterized clonal sectors derived from genetic reversion of rolC (A. rhizogenes)--induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S. cerevisiae, in tobacco. The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers. Two transgenic male-sterile tobacco lines (N. tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line. Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F1 (FLP x FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras. These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.

Effect of polyethylene glycol on rumen volume and retention time of liquid and particulate matter along the digestive tract in goats fed tannin-rich carob leaves (Ceratonia siliqua).

The present work studied the effects of tannins in carob leaves (CL) on rumen volume and kinetics, and on the retention time of fluid and particulate components of the digesta along the gastrointestinal tract (GIT) in goats. The experimental design was a two factor crossover experiment, i.e. in phase 1, two goats were fed CL and 2 CL and polyethylene glycol (PEG) and in phase 2, the treatments were switched. The main effects of tannins were depression of the rumen fluid and particulate content of the rumen, acceleration of the passage of liquid from the abomasum, and delay of the passage of digesta in the intestine. The overall effect was a delay in the passage of fluid and particulate matter throughout the entire GIT. It is hypothesised that these responses are largely the consequence of the interaction of tannins with digestive enzymes and the epithelium lining of the digestive tract.

Pediatric renal transplantation under tacrolimus-based immunosuppression.

BACKGROUND: Tacrolimus has been used as a primary immunosuppressive agent in adult and pediatric renal transplant recipients, with reasonable outcomes. Methods. Between December 14, 1989 and December 31, 1996, 82 pediatric renal transplantations alone were performed under tacrolimus-based immunosuppression without induction anti-lymphocyte antibody therapy. Patients undergoing concomitant or prior liver and/or intestinal transplantation were not included in the analysis. The mean recipient age was 10.6+/-5.2 years (range: 0.7-17.9). Eighteen (22%) cases were repeat transplantations, and 6 (7%) were in patients with panel-reactive antibody levels over 40%. Thirty-four (41%) cases were with living donors, and 48 (59%) were with cadaveric donors. The mean donor age was 27.3+/-14.6 years (range: 0.7-50), and the mean cold ischemia time in the cadaveric cases was 26.5+/-8.8 hr. The mean number of HLA matches and mismatches was 2.8+/-1.2 and 2.9+/-1.3; there were five (6%) O-Ag mismatches. The mean follow-up was 4.0+/-0.2 years. RESULTS: The 1- and 4-year actuarial patient survival was 99% and 94%. The 1- and 4-year actuarial graft survival was 98% and 84%. The mean serum creatinine was 1.1+/-0.5 mg/dl, and the corresponding calculated creatinine clearance was 88+/-25 ml/min/1.73 m2. A total of 66% of successfully transplanted patients were withdrawn from prednisone. In children who were withdrawn from steroids, the mean standard deviation height scores (Z-score) at the time of transplantation and at 1 and 4 years were -2.3+/-2.0, -1.7+/-1.0, and +0.36+/-1.5. Eighty-six percent of successfully transplanted patients were not taking anti-hypertensive medications. The incidence of acute rejection was 44%; between December 1989 and December 1993, it was 63%, and between January 1994 and December 1996, it was 23% (P=0.0003). The incidence of steroid-resistant rejection was 5%. The incidence of delayed graft function was 5%, and 2% of patients required dialysis within 1 week of transplantation. The incidence of cytomegalovirus was 13%; between December 1989 and December 1992, it was 17%, and between January 1993 and December 1996, it was 12%. The incidence of early Epstein-Barr virus-related posttransplant lymphoproliferative disorder (PTLD) was 9%; between December 1989 and December 1992, it was 17%, and between January 1993 and December 1996, it was 4%. All of the early PTLD cases were treated successfully with temporary cessation of immunosuppression and institution of antiviral therapy, without patient or graft loss. CONCLUSIONS: These data demonstrate the short- and medium-term efficacy of tacrolimus-based immunosuppression in pediatric renal transplant recipients, with reasonable patient and graft survival, routine achievement of steroid and anti-hypertensive medication withdrawal, gratifying increases in growth, and, with further experience, a decreasing incidence of both rejection and PTLD.

Tacrolimus in pediatric renal transplantation.

Tacrolimus was used as the primary immunosuppressive agent in 69 pediatric renal transplantations between December 17, 1989, and June 30, 1995. Children undergoing concomitant or prior liver and/or intestinal transplantation were excluded from analysis. The mean recipient age was 10.3+/-5.0 years (range, 0.7-17.5 years). Seventeen (24.6%) children were undergoing retransplantation, and six (8.7%) had a panel reactive antibody level of 40% or higher. Thirty-nine (57%) cases were with cadaveric kidneys, and 30 (43%) were with living donors. The mean donor age was 28.0+/-14.7 years (range, 1.0-50.0 years), and the mean cold ischemia time for the cadaveric kidneys was 27.0+/-9.4 hr. The antigen match was 2.7+/-1.2, and the mismatch was 3.1+/-1.2. All patients received tacrolimus and steroids, without antibody induction, and 26% received azathioprine as well. The mean follow-up was 32+/-20 months. One- and 4-year actuarial patient survival rates were 100% and 95%. One- and 4-year actuarial graft survival rates were 99% and 85%. The mean serum creatinine level was 1.2+/-0.8 mg/dl, and the calculated creatinine clearance was 82+/-26 ml/min/1.73 m2. The mean tacrolimus dose was 0.22+/-0.14 mg/ kg/day, and the level was 9.5+/-4.8 ng/ml. The mean prednisone dose was 2.1+/-4.9 mg/day (0.07+/-0.17 mg/kg/day), and 73% of successfully transplanted children were off prednisone. Seventy-nine percent were not taking any antihypertensive medications. The mean serum cholesterol level was 158+/-54 mg/dl. The incidence of delayed graft function was 4.3%. The incidence of rejection was 49%, and the incidence of steroid-resistant rejection was 6%. The incidence of rejection decreased to 27% in the most recent 26 cases (January 1994 through June 1995). The incidence of new-onset diabetes was 10.1%; six of the seven affected children were able to be weaned off insulin. The incidence of cytomegalovirus disease was 13%, and that of posttransplant lymphoproliferative disorder was 10%; the incidence of posttransplant lymphoproliferative disorder in the last 40 transplants was 5% (two cases). All of the children who developed posttransplant lymphoproliferative disorder are alive and have functioning allografts. Based on this data, we believe that tacrolimus is a superior immunosuppressive agent in pediatric renal transplant patients, with excellent short- and medium-term patient and graft survival, an ability to withdraw steroids in the majority of patients, and, with more experience, a decreasing rate of rejection and viral complications.

The use of tacrolimus in renal transplantation.

Tacrolimus (FK 506) is a novel immunosuppressive agent that has been in clinical use for solid organ transplantation since 1989. Early clinical trials of tacrolimus in liver, heart, kidney, lung, and intestinal transplantation at the University of Pittsburgh have demonstrated it to be a safe and effective agent with several potential advantages over existing immunosuppressive drugs. More recently, phase I and II multicenter trials of tacrolimus for renal transplantation have been performed; however, data are not yet available from these trials. Our experience with this drug has demonstrated excellent 1- and 2-year actuarial graft survival rates of 89% and 83%, respectively, in adult renal transplantation and 1- and 3-year graft survival rates of 98% and 85%, respectively in pediatric renal transplantation. A major advantage of tacrolimus noted in these trials was the ability to discontinue steroid therapy in approximately 50% of the patients. Tacrolimus has also shown efficacy as a rescue agent for renal allograft rejection failing conventional therapy in 74% of cases. This paper expands on these observations and focuses on the experience we have gained with the use of tacrolimus at our institution over the last 6 years.

Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase.

FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of ß-glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.

The superiority of tacrolimus in renal transplant recipients -- the Pittsburgh experience.

Tacrolimus is a superior immunosuppressive agent in patients undergoing renal transplantation. In adults, the 1- and 3-year actuarial patient survivals were 95% and 92%, and the 1- and 3-year actuarial graft survivals were 89% and 80%. For first cadaver kidneys, the 1- and 3-year actuarial graft survivals were 91% and 82%, with a projected half-life of 11.9 years. Sixty-nine percent of successfully transplanted patients were weaned off steroids. In pediatric patients, the 1- and 4-year actuarial patient survivals were 100% and 96%, and the 1- and 4-year actuarial graft survivals were 99% and 85%. Seventy-three percent of successfully transplanted children were weaned off steroids. Tacrolimus was also useful as a rescue agent, with an initial success rate of 74%. Tacrolimus has been used successfully in kidney/ pancreas transplantation, with 100% patient, 95% kidney, and 79% pancreas graft survival. Tacrolimus should be considered the immunosuppressive agent of choice in renal transplantation.

FK506 in pediatric kidney transplantation--primary and rescue experience.

Between 14 December 1989 and 17 December 1993, 43 patients undergoing kidney transplantation alone at the Children's Hospital of Pittsburgh received FK506 as the primary immunosuppressive agent. The mean recipient age was 10.2 +/- 4.8 years (range 0.7-17.4 years), with 7 (16%) children under 5 years of age and 2 (5%) under 2 years of age. Fifteen (35%) children underwent retransplantation, and 5 (12%) had a panel-reactive antibody level greater than 40%. Twenty-two (51%) transplants were with cadaveric donors and 21 (49%) were with living donors. The mean follow-up was 25 +/- 14 months; there were no deaths; 1- and 3-year actuarial graft survival was 98% and 85%. The mean serum creatinine and blood urea nitrogen were 1.2 +/- 0.6 mg/dl and 26 +/- 11 mg/dl; the calculated creatinine clearance was 75 +/- 23 ml/min per 1.73 m2. Twenty-four (62%) patients have been successfully withdrawn from steroids and 24 (62%) require no anti-hypertensive medication. Improved growth was seen, particularly in pre-adolescent children off steroids. Between 28 July 1990 and 2 December 1993, 24 children were referred for rescue therapy with FK506, 14.6 +/- 16.4 months (range 1.1-53.2 months) after transplantation. Nineteen (79%) were referred because of resistant rejection; 4 (17%) were referred because of proteinuria; 1 (4%) was switched because of steroid-related obesity. There were no deaths; 1- and 2-year graft survival was 75% and 68%; 17 (71%) patients were successfully rescued, including 1 of 2 patients who arrived on dialysis; 4 (24%) of the successfully rescued patients were weaned off steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Improving results of pediatric renal transplantation.

BACKGROUND: Outcome after renal transplantation in children has been variable. We undertook a retrospective study of our experience over the past five years. STUDY DESIGN: From January 1, 1988, to October 15, 1992, 60 renal transplantations were performed upon 59 children at the Children's Hospital of Pittsburgh. Twenty-eight (47 percent) of the kidneys were from cadaveric donors, and 32 (53 percent) were from living donors. The recipients ranged in age from 0.8 to 17.4 years, with a mean of 9.8 +/- 4.8 years. Forty-six (77 percent) recipients were undergoing a first transplant, while 14 (23 percent) received a second or third transplant. Eight (13 percent) of the patients were sensitized, with a panel reactive antibody of more than 40 percent. Eleven of the 14 patients undergoing retransplantation and seven of the eight patients who were sensitized received kidneys from cadaveric donors. Thirty-three (55 percent) patients received cyclosporine-based immunosuppression, and 27 (45 percent) received FK506 as the primary immunosuppressive agent. RESULTS: The median follow-up period was 36 months, with a range of six to 63 months. The one- and four-year actuarial patient survival rate was 100 and 98 percent. The one- and four-year actuarial graft survival rate was 98 and 83 percent. For living donor recipients, the one- and four-year actuarial patient survival rate was 100 and 100 percent; for cadaveric recipients, it was 100 and 96 percent. Corresponding one- and four-year actuarial graft survival rates were 100 and 95 percent for the living donor recipients and 96 and 69 percent for the cadaveric recipients. Patients on cyclosporine had a one- and four-year patient survival rate of 100 and 97 percent, and patients on FK506 had a one- and three-year patient survival rate of 100 and 100 percent. Corresponding one- and four-year actuarial graft survival rates were 100 and 85 percent in the cyclosporine group, while one- and three-year actuarial graft survival rates were 96 and 84 percent in the FK506 group. The mean serum creatinine level was 1.24 +/- 0.64 mg per dL; the blood urea nitrogen level was 26 +/- 13 mg per dL. The incidence of rejection was 47 percent; 75 percent of the rejections were steroid-responsive. The incidence of cytomegalovirus was 10 percent. The incidence of post-transplant lymphoproliferative disorder was 8 percent. None of the patients on cyclosporine were able to be taken off prednisone; 56 percent of the patients receiving FK506 were taken off prednisone successfully. Early growth and development data suggest that the patients receiving FK506 off prednisone had significant gains in growth. CONCLUSIONS: These results support the idea that renal transplantation is a successful therapy for end-stage renal disease in children. They also illustrate the potential benefits of a new immunosuppressive agent, FK506.

Acute myocardial infarction in a young boy with nephrotic syndrome: a case report and review of the literature.

A 7-year-old boy with a 5-year history of steroid-unresponsive nephrotic syndrome due to minimal change disease presented with acute myocardial infarction. Angiography was suggestive of a dissected atherosclerotic plaque at the initial and mid portions of the right coronary artery, as well as a lesion in the mid portion of the circumflex artery. The child had a long history of extreme hypercholesterolemia and hypertriglyceridemia, along with apolipoprotein-E 4/3 phenotype. The mother, who also has apolipoprotein-E 4/3 phenotype, has mild hypercholesterolemia. The case suggests that children with long-lasting nephrotic syndrome and even mild familial propensity for hyperlipidemia may be at increased risk for ischemic cardiovascular events. The literature is reviewed regarding the relationship between nephrotic syndrome and the incidence of ischemic heart disease.

Comparison of FK-506 and cyclosporine regimens in pediatric renal transplantation.

Clinical aspects of FK-506 or cyclosporine immunosuppression regimens were evaluated in 48 consecutive pediatric renal transplant recipients. Tapering and discontinuation of prednisone was employed only in children receiving FK-506 who experienced minor or no rejection episodes during the 1st posttransplant month. At 1 year follow-up, 17 of 22 (77%) of all children with functioning allografts were receiving no prednisone (n = 13) or a mean dosage of 0.07 mg/kg per day (n = 4). During the 1st month, acute cellular rejection was more common in the FK-506 group (0.58 vs. 0.21 rejections per patient, P < 0.05) but allograft survival (92%) and renal function at 1 year posttransplant were identical in both groups. Compared with the cyclosporine regimen, FK-506 immunosuppression may be associated with a higher incidence of cytomegalovirus or reversible Epstein-Barr virus-induced lymphoproliferative disease. However, the FK-506 group had less hirsutism and gingival hypertrophy and required fewer antihypertensive medications independent of steroid use. Height standard deviation scores and weight-for-height index improved only in pre-adolescents receiving FK-506 but no prednisone (P < 0.02 and P < 0.05, respectively), but did not differ between children on FK-506 plus prednisone and those in the cyclosporine group. We conclude that the major advantages of FK-506 over cyclosporine immunosuppression are a reduced severity of hypertension and an improved cosmetic appearance which may improve long-term medical compliance. When used as monotherapy, FK-506 also shows promise in relieving the growth retardation associated with cyclosporine regimens that include prednisone.

Acute noncrescentic poststreptococcal glomerulonephritis presenting with pulmonary hemorrhage.

We report a child with acute poststreptococcal noncrescentic glomerulonephritis and pulmonary hemorrhage. This patient demonstrates that: (1) poststreptococcal noncrescentic glomerulonephritis in children can be associated with pulmonary hemorrhage, (2) an expeditious renal biopsy in patients with acute or rapidly progressive glomerulonephritis and pulmonary hemorrhage can establish an early diagnosis and provide timely guidance for treatment, and (3) although not proven by controlled studies, the intravenous administration of methylprednisolone in our patient may have been effective in the treatment of pulmonary hemorrhage.

IgA nephritis in a patient with Alagille syndrome and a transplanted liver.

Alagille syndrome (arteriohepatic dysplasia) is a major cause of intrahepatic cholestasis in infancy. The present report describes a patient with Alagille syndrome who presented with hematuria and IgA nephritis 7 years after an orthotopic liver transplantation and immunosuppression. This patient suggests that glomerular lipidosis is not an inherent feature of the Alagille syndrome, and that IgA nephritis may develop in spite of ongoing immunosuppressive treatment.

Urokinase in conditioned medium from phorbol ester-pretreated endothelial cells promotes polymorphonuclear leukocyte migration.

Serum-free conditioned medium (CM) generated by human umbilical vein endothelial cell monolayers following pretreatment with 100 ng/ml of phorbol myristate acetate (PMA) promoted human polymorphonuclear leukocyte (PMNL) migration as assayed in blindwell chambers. Stimulation of PMNL migration in response to CM was dependent on the dose of PMA used to pretreat the endothelial cells as well as the duration of incubation time to generate CM. Phorbol esters have been previously shown to release plasminogen activators from vascular endothelial cells. In the present study, pretreatment of endothelial cells with PMA also increased plasminogen activator activity in CM at a time course similar to the generation of PMNL chemoattractant activity. Treatment of CM with a polyclonal antibody against human urokinase-type plasminogen activator (uPA) not only inhibited uPA activity, but also significantly reduced PMNL chemoattractant activity when compared with untreated CM. In contrast, treatment of CM with an antibody directed against tissue-type plasminogen activator (tPA) did not affect PMNL migratory activity. Furthermore, when CM was passed over an anti-uPA immunoaffinity column, plasminogen activator activity was reduced 90% and chemoattractant activities was reduced 68%. Both plasminogen activator and chemoattractant activities were reconstituted in the eluate from the anti-uPA column. These data demonstrate that uPA present in the CM from PMA-pretreated endothelial cells stimulates PMNL chemoattractant activity and suggests a possible role for endothelial cell-derived uPA in stimulating migration of peripheral blood leukocytes at an inflammatory locus.