Fr van der Leij

Molecular cloning and partial characterization of the gene for granule-bound starch synthase from a wildtype and an amylose-free potato (Solanum tuberosum L.)

Abstract The gene encoding granule-bound starch synthase (GBSS), which determines the presence of amylose in reserve starches, has been isolated from a wild-type and from an amylose-free potato by using a potato GBSS cDNA. From the analysis of five genomic GBSS clones, isolated from the wild-type potato, it is shown that GBSS is a single copy gene in potato. GBSS messenger RNA was shown to be present in a number of different tissues, but was most abundantly found in stolons and tubers. Southern blot analysis of the GBSS genes from both potato genotypes revealed that the amylose-free mutant, which lacks GBSS activity and protein, does not contain a large structural lesion in the GBSS gene. The GBSS messenger RNA was even found to be present far more abundant in the mutant than in the wild-type potato.

Alterations in adult rat heart after neonatal dexamethasone therapy

Glucocorticoid treatment in preterm babies to prevent chronic lung disease causes myocardial hypertrophy and increased myocardial protein content. Although these changes are thought to be transient, there is evidence that dexamethasone (DEX) induces permanent myocardial abnormalities as well. We investigated whether a therapeutic course of neonatal DEX in rat pups produces anatomic and biochemical alterations in rat hearts during adult life. Twenty-four rat pups were treated with DEX on d 1, 2, and 3 (0.5, 0.3, and 0.1 μg/g) of life, with doses proportional to those used in preterm babies. Twenty-four control pups were treated with saline. At d 7, wk 8, or wk 45 (n = 8 per group) rats were killed. The anatomic parameters measured were body weight (Bw, in grams), heart (myocardial) weight (Hw, in milligrams), and the Hw:Bw ratio. Myocardial total protein (Prot) and DNA content were determined, and the Prot:DNA ratio was calculated. Histopathology and morphometry were performed on 45-wk-old rat hearts. In DEX-treated rat pups, at d 7, Bw and Hw were lower and the Hw:Bw ratio was increased. DNA content was lower, Prot higher, and Prot:DNA ratio was increased. In 8-wk-old rats Bw, Hw, DNA content, Prot content or Prot:DNA ratio did not differ between groups, but the Prot:DNA ratio still tended to be higher in DEX-treated rats. In 45-wk-old rats Hw and Hw:Bw ratio were significantly lower and Prot:DNA ratio higher in DEX-treated rats. Histopathologic analysis showed larger cardiomyocyte volume, length, and width, indicating hypertrophy, and increased collagen, indicating early degeneration of individual myocytes. In conclusion, neonatal DEX treatment in rat pups causes a permanent decrease in heart weight, as well as hypertrophy and early degeneration of cardiomyocytes during adulthood.

In situ hybridization to somatic metaphase chromosomes of potato

SummaryAn in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a “ladderpattern” in genomic Southerns of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.

Suppression of physiological cardiomyocyte proliferation in the rat pup after neonatal glucocorticosteroid treatment

AbstractBackgroundGlucocorticosteroids (mostly dexamethasone) are widely used to prevent chronic lung disease in premature infants. Neonatal rats treated with dexamethasone have been shown to have reduced cardiac mass and cardiomyocyte hypertrophy, suggesting a lower number of cardiomyocytes at adult age, and a severely reduced life expectancy. In the present study we tested the hypothesis that a lower number of cardiomyocytes in later life is caused by a reduced cardiomyocyte proliferation and/or by early cell death (apoptosis).Methods and resultsRat pups received dexamethasone or saline control on day 1, 2 and 3 and were sacrificed at day 0, 2, 4, 7 and 21. The cardiomyocytes of dexamethasone treated pups showed a reduced proliferation as indicated by a lower mitotic index and reduced number of Ki–67 positive cardiomyocytes on day 2 and 4 as compared to day 0 and day 7 and also as compared to the age–matched saline pups. On day 7 and day 21 the mitotic index was not different between groups. From day 2 onward up to day 21 dexamethasone treated pups showed a lower number of cardiomyocytes. The cardiomyocytes showed no signs (<<1%) of apoptosis (Caspase–3 and cleaved–PARP) in any group.ConclusionThe temporary suppression of cardiomyocyte hyperplasia found in dexamethasone treated pups eventually leads to a reduced number and hypertrophy of cardiomyocytes during adult life.

Chimerism as the basis for the occurrence of amylose synthesizing clones derived from an amylose-free potato mutant

Earlier described revertants, obtained after irradiation of an amylose-free (amf) mutant which carries a point deletion in the gene for granule-bound starch synthase, were analysed at the DNA-sequence level. Direct sequencing of fragments amplified by the polymerase chain reaction revealed that all investigated revertants carry the original wildtype sequence. It is argued that mutation as the basis for the re-occurrence of wildtype alleles is highly unlikely. The alternative conclusion is reached that the original amylose-free monoploid clone must have been a chimera. Chimerism with wildtype and mutant tissues was actually found in a plant which at a later stage was obtained from the same mutant clone without the use of X-rays. Wildtype cells could have remained in the L2 layer of the original monoploid mutant, which cannot be analyzed for starch composition.

Confocal Laser Scanning Microscopy of Human Skin Fibroblasts Showing Transient Expression of a Green Fluorescent Carnitine Palmitoyltransferase 1 Fusion Protein

The mitochondrial outer membrane enzyme carnitine palmitoyltransferase 1 (CPT1) is a main site of regulation of intracellular long-chain fatty acid transport. At least two isoforms of CPT1 are expressed in the body: L-CPT1 (the “liver-type” isoform) and M-CPT1 (the “muscle-type” isoform). Skin fibroblasts from healthy humans are known to contain only one isoform of CPT1: the liver-type, which is encoded by the gene CPT1A. Skin fibroblasts from patients with a liver-type CPT1 deficiency do not express either of the two known CPT1 isoforms (neither livernor muscle-type), and therefore could provide an excellent background to study CPT1 by means of molecular complementation. In this chapter, we describe the first experiments we carried out with a gene fusion of a complementary DNA of the human gene for muscle-type carnitine palmitoyltransferase (CPT1B) and a gene encoding an “enhanced” green fluorescent protein (GFP). We wished to express the human CPT1B gene in human skin fibroblasts, taking the following facts into consideration: