Cystic fibrosis gene mutations and linked RFLPs in the Slovenian population.

The authors used polymerase chain reaction to analyse 56 Slovenian cystic fibrosis (CF) chromosomes for the presence of delta F508 and eight other most frequent mutations located in exons 7,11 and 20 (R347P, R334W, G551D, R553X, S549RA, S549RT, S549I and S1255X) of the CF gene. We also determined the frequency of haplotypes associated with CF for six linked RFLP markers (MetD/TaqI, MetH/TaqI, XV-2c/TaqI, KM-19/PstI, MP6d9/MspI and J3.11/MspI) in 27 Slovenian CF families. delta F508 mutation was present in 55.4 percent of the CF chromosomes. No case of the other mutations were detected in the sample of tested CF chromosomes. A very high degree of association (0.88) has been found between DNA marker MetH and CF (as measured by the Yule's association coefficient) in our population. Using the RFLP markers XV-2c and KM-19, we found that 85% of delta F508 mutated chromosomes have a single 1 2 (B) haplotype, and that this haplotype is present on only 15.4 percent of CF chromosomes without this deletion.     

Detection of complex alleles by direct analysis of DNA heteroduplexes

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.     

Growth hormone releasing effect of hpGRF-40 in rats at different time intervals following ablation of the mediobasal hypothalamus

We have investigated the effect of hypothalamo-pituitary disconnection in the rat on the growth hormone (GH) responsiveness to human pancreatic GH-releasing factor (hpGRF). Adult female rats, sham-operated (sham-op) or bearing a complete mechanical ablation of the mediobasal hypothalamus (MBH-A) were challenged, while under urethane anesthesia, with hpGRF-40 (20,100,500 ng/rat i.v.) at different time intervals after surgery. In sham-op rats only 500 ng/rat of hpGRF-40 stimulated GH release, while in 1-and 7-day MBH-A rats the stimulation also occurred with the lower hpGRF doses and the rise in plasma GH was greater than in sham-op controls. Twenty-one and 42 days after the placing of the lesions the GH response to hpGRF-40 was still present at the 500 ng/rat dose, though it was smaller than in sham-op controls. Evaluation of pituitary GH content demonstrated a progressive and rapid decline starting the first day after the placing of the lesions. These data indicate that GH responsiveness to hpGRF is: 1) enhanced in the anterior pituitary shortly after hypothalamo-pituitary disconnection and, 2) despite a striking reduction of the pituitary GH stores, it is maintained after these lesions.The physiologic growth hormone (GH) releaser in the rat is GH-releasing factor and, recently, a group of peptides has been characterized from human pancreatic tumors (hpGRFs) (1,2) which are potent and specific GH-releasers in both animals (3) and man (4). The availability of these peptides, which show a high degree of homology with the physiologic rat hypothalamic GRF (5), offers the unique opportunity to assess somatotrope responsiveness to GRF molecules in rats with hypothalamo-pituitary disconnection.In this study we have first evaluated the GH pituitary responsiveness to increasing doses of hpGRF-40 in rats following mechanical ablation of the mediobasal hypothalamus (6). These rats, by definition, lack the effect of both central nervous system (CNS) inhibitory (e.g. somatostatin) and stimulatory (e.g. GRF) influences to GH release. With the aim to ascertain how the lack of these two opposing inputs reflects on the secretory capacity of the somatotropes, we also investigated the GH response to hpGRF-40 at different time intervals after the lesioning. In a study in rats with electrolytic lesions of the ventromedial-arcuate region of the hypothalamus Tannenbaum et al (7) had shown persistence of the GH response to huge doses of a hpGRF analog.     

Die Bedeutung der Familien für die Bearbeitung pflanzensoziologischer Tabellen

Zusammenfassung Die Häufigkeit des Vorkommens einzelner Familien in Vegetationstabellen scheint einer Gesetzmäßigkeit zu unterliegen. Bei Pioniergesellschaften kommt es vor, daß eine Familie mit weitaus überwiegender Frequenz aufscheint, während klimaxnahe Gesellschaften ein besseres Gleichgewicht der Familienanteile aufzuweisen scheinen.

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Summary The frequency of representative species of families in phytosociological tables seems to depend on a general law. Pioneer associations present a family that is largely dominating, while associations near to climax show a better equilibrium.