Biomedical subjects

M Tuyl

Publications and source records attributed to M Tuyl.

3 recordsLinked to original sources

PubMed1990

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.

Blotting, Northern↗

PubMedDate not supplied

Calmodulin inhibitors potentiate hyperthermic cell killing.

The role of calmodulin (CaM) in cellular heat injury of neuroblastoma N2A and hepatoma H35 cells has been investigated, using specific calmodulin-inhibiting drugs (Trifluoperazine, Compound 48/80 and Calmidazolium). These CaM-specific drugs potentiate hyperthermia-induced cell killing, suggesting CaM to be involved in processes aimed on the repair of heat injury. The CaM inhibitors also prevent hyperthermia-induced cytoskeletal alterations in the cell types studied. The action of CaM inhibitors was dose dependent, and seems to be confined to the first period of the hyperthermic treatment. Neither production of heat shock proteins in heat-shocked cultures, nor the rate of protein synthesis in control cultures were affected by the CaM inhibitors. It was concluded that an inverse correlation exists between hyperthermic cell killing and cytoskeletal alterations. Activation of CaM is suggested to be a fundamental aspect of the cellular heat shock response.

Animals↗

PubMedDate not supplied

Evidence for two states of thermotolerance.

The phenomenon of thermotolerance has been studied in three-day-old larvae of Lymnaea stagnalis (Pulmonata, Mollusca). The lethality of a test treatment (TT) of 1 h, 40.0 degrees C is greatly reduced by pretreatment at elevated temperatures. The induced thermotolerance shows characteristics which are dependent on duration and temperature of the pretreatment (conditioning treatment; CT). Thermotolerance induced by a CT of 1 h at greater than 38.0 degrees C is stable at least during 2 h at 25 degrees C, whereas thermotolerance induced by the lower temperatures decays partly. Five minute CTs at 33 and 35 degrees C induce thermotolerance as well; however, this fades completely within 1 h at 25 degrees C. The same level of tolerance is present after a 5 min CT at 38 degrees C, but this level continues to rise during 1 h at 25 degrees C. The induction of thermotolerance (CT 1 h) is suppressed only partly when protein synthesis is inhibited by puromycin. The effect of puromycin is relatively weaker at the lower conditioning temperatures. Furthermore, puromycin slows down the decay of thermotolerance. The influence of duration and temperature of heat treatment on the pattern of protein synthesis has been investigated by gel electrophoresis after incubation in [35S]methionine. Heat-shock proteins (hsp) are synthesized during 1 h incubation at 35, 37, and 38 degrees C, but not at 33 degrees C. Twenty-four hours at 33 degrees C does induce the synthesis of hsp. It is proposed that thermotolerance in Lymnaea is based on two states, a rather unstable one which is independent of hsp synthesis, and another more stable one, accompanied by hsp synthesis.

Adaptation, Physiological↗