[Magnesium deficiency and therapy in cardiac arrhythmias: recommendations of the German Society for Magnesium Research]. / Magnesiummangel und Magnesiumtherapie bei Herzrhythmusstörungen. Empfehlungen der Gesellschaft für Magnesium-Forschung e. V.

Aim of the recommendations of the German Society for Magnesium Research: Recognition and compensation of magnesium deficiency in patients with risk factors for cardiac arrhythmias or manifest rhythm disturbances. Prevention of arrhythmias by administration of magnesium. Therapeutic administration of magnesium in patients with arrhythmias with and without magnesium deficiency. The current state of knowledge claims for considering the status of magnesium and the possibility of a therapeutic intervention with magnesium within the concept of the treatment of cardiovascular diseases. The use of magnesium as single agent or as an adjunct to other therapeutic actions in the prevention and therapy of cardiac arrhythmias can be effective and, in case of oral administration, very safe. In case of parenteral administration, it is important to use adequate doses, monitor cardiovascular and neuromuscular parameters and to consider contraindications.

Subcloning of prochymosin cDNA for Plac controlled expression.

Two overlapping segments of prochymosin cDNA clones (Liebscher et al., 1985) were used to construct plasmids that expressed an activable zymogen product and thus verified the integrity of the reverse transcripts. The pUC9 vector was used for the expression, under the control of the lac promoter. The expression product (a fused protein consisting of the N-terminus of beta-galactosidase, a polylinker-coded peptide and prochymosin from its 5th amino acid) displayed, upon activation by the usual procedure, the properties of calf chymosin. The active product was identified by milk-clotting tests, "caseinography" and protein electrophoresis of immunoprecipitates. The "boxing" of prochymosin cDNA in the constructed plasmids makes them a versatile source of this cDNA for other expression constructs.

Molecular cloning and identification of cDNA recombinants coding for bovine prochymosin in E. coli.

Poly(A) RNA was isolated from the gastric mucosa of the bovine fourth stomach (the abomasum) using and analysing several calves not older than 12 days. The amount of the preprochymosin mRNA in the mucosa of those animals at best reaches about 5-10% of the poly(A) RNA as estimated by in vitro translation and immunoprecipitation. Starting from that material double-stranded complementary DNA was synthesized, inserted by dG dC tailing into the PstI site of the vector plasmid pBR322 and used for transformation of E. coli. Tetracycline resistant clones containing DNA sequences coding for the full length of prochymosin were recognized by colony hybridization with five specific d-oligonucleotides corresponding either to the N-terminal, the middle or the C-terminal part of prochymosin. Six recombinants were detected by screening of 1 500 recombinants with an oligonucleotide which corresponds to positions 649 to 663 of the nucleotide sequence published by Harris et al. (1982). Two of them were found to cover together the complete prochymosin sequence as evidenced by both positive colony hybridization with either the N-terminal or the C-terminal oligonucleotide probe, as well as by the restriction pattern of the selected plasmids.

Carp preproinsulin cDNA sequence and evolution of insulin genes.

The nucleic acid sequence of the preproinsulin cDNA of carp (Cyprinus carpio), cloned in the PstI site of pBR322 ( Liebscher et al. 1980), has been determined. The sequenced insert of 439 bp includes the complete coding information for carp preproinsulin (108 amino acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'-nontranslated regions. The nucleotide sequence confirms the previously established amino acid sequence of carp insulin ( Makower et al. 1982) and determines those of the signal 21 amino acids and C peptide (35 amino acids). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp insulin B chain suggest that the cleavage site of the signal peptidase has moved. Calculations based on the comparison of known preproinsulin cDNA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than that between man and chicken.

Carp preproinsulin cDNA sequence and evolution of insulin genes.

The nucleic acid sequence of the preproinsulin cDNA of carp (Cyprinus carpio), cloned in the PstI-site of pBR322 (1), has been determined. The sequenced insert of 439 bp includes the complete coding information for carp preproinsulin (108 amino acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'-nontranslated regions. The nucleotide sequence confirms the previously established amino acid sequence of carp insulin (2) and determines those of the signal (21 aa 1) and C-peptide (35 aa 1). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp insulin B-chain suggest that the cleavage site of the signal peptidase has moved. Calculations based on the comparison of known preproinsulin cDNA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than between man and chicken.

Isolation and characterization of human liver mRNA.

Poly(A)-RNA isolated from human liver shows a predominant size in the 18 S region. It can be translated into a broad range of proteins up to molecular weights of more than 100 000 with predominant translation products in the 45 000-55 000 region. Full length cDNA has been transcribed and used for a complexity analysis of this mRNA giving a total sequence complexity of about 7 000 different mRNA species.

Cloning of carp preproinsulin cDNA in the bacterial plasmid pBR322.

The successful cloning of recombinants between cDNA from fractionated poly(A)+-RNA of Brockmann bodies of the carp and the plasmid pBR322 in Escherichia coli chi 1776 is reported. One of the recombinant clones has been identified as a preproinsulin-cDNA recombinant by the hybrid-arrest translation assay. Recombination was at the PstI site of pBR322; reconstitution of this site was by 3'-tailing of the vector with dGn. The transformants were screened by in situ hybridization with kinase-labeled poly(A)+-RNA sedimenting at 9S from Brockmann bodies. Restriction analysis was performed on 26 of the strongly hybridizing clones to estimate the size of the inserted cDNA. Six of the recombinants studied contain inserts of a size approximating to full length 9S preproinsulin mRNA. The hybrid-arrest translation assay on selected clones identified one as a recombinant containing the preproinsulin cDNA sequence.

In vivo and in vitro recombination of lambda DNA in CaC12 transfection.

Using CaCl2 mediated transfection with Lambda DNA fragments, in vitro joining by ligase and in vivo recombination with helper phage DNA are effective systems for generating artificial recombinants. Recombination efficiencies are 20--30% in the in vitro and in vivo recombination systems. At 30 to 37 degree C T4 ligase mainly joins natural cohesive alpha ends, while at 12 degrees C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector alpha 401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCl2 transfection. For in vivo recombination a new CaCl2 transfection system was developed, termed postinfection-dependent CaCl2 transfection system, which is based on the infection of recipient cells with helper phages after transfection. In marker rescue experiments using this method not only single but also double recombination occurred between two independent alpha DNA fragments and the helper phage DNA.

Factors which influence CaC12 dependent transfection of lambda DNA in Escherichia coli K12 recipients.

This paper presents further parameters influencing the competence, the process of DNA uptake and the efficiency of plating of CaC12-treated E. coli D12 strains. We have found that the process of DNA uptake depends not only on the treatment of bacteria with a certain CaCl2-concentration but is also influenced considerably by a shift-down of the CaCl2-concentration in the reaction mixture. The pH of the growth media and of the reaction mixture plays an important role in maintaining of optimal transfection. The efficiency of plating is influenced by the thickness of the top layer and the concentration of bacteria on the plate. Without genetic variation of the strains, by only varying the mentioned factors we could improve the efficiency of CaCl2 transfection at about two orders of magnitude to a maximum of 6 X 105 pfu/microgram DNA.