ABSTRACT
Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 × 103 and 3.3 × 108 viral genome copies with a linearity of R 2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.
ABSTRACT
The effect of in vitro addition of zinc sulphate on T4 deiodination in brown adipose tissue (BAT) of rats exposed to 4 degrees C or 22 degrees C temperature during 24 h, was studied. Animals were killed by cervical dislocation and BAT was immediately removed and homogenized in sucrose buffer (320 mM) containing HEPES (10 mM) pH 7.4. The preparation was centrifuged at 4 degrees C during 10 min. Aliquots were separated adding 50, 100 microM, 1 o 5 mM zinc sulphate plus 0, 5, 10 or 25 mM dithiothreitol plus 1 microCi 125I-T4. The mixture was incubated at 37 degrees C during 60 min. Aliquots were applied to Whatman paper and chromatographed. BAT from control rats kept at 22 degrees C produced 79 +/- 30 pg T3/g protein/h. This value was significantly reduced in homogenates containing 1 or 5 mM zinc. In rats exposed to 4 degrees C, T3 production increased to 248 +/- 37 pg/mg protein/h. The addition of 100 microM, 1 or 5 mM zinc significantly decreased T3 production. The inhibitory action of zinc on BAT T4 deiodination may have a deleterious effect on BAT thermogenesis.
Subject(s)
Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Thyroxine/metabolism , Triiodothyronine/antagonists & inhibitors , Triiodothyronine/metabolism , Zinc Sulfate/pharmacology , Animals , In Vitro Techniques , Rats , Rats, Wistar , TemperatureABSTRACT
The effect of in vitro addition of zinc sulphate on T4 deiodination in brown adipose tissue (BAT) of rats exposed to 4 degrees C or 22 degrees C temperature during 24 h, was studied. Animals were killed by cervical dislocation and BAT was immediately removed and homogenized in sucrose buffer (320 mM) containing HEPES (10 mM) pH 7.4. The preparation was centrifuged at 4 degrees C during 10 min. Aliquots were separated adding 50, 100 microM, 1 o 5 mM zinc sulphate plus 0, 5, 10 or 25 mM dithiothreitol plus 1 microCi 125I-T4. The mixture was incubated at 37 degrees C during 60 min. Aliquots were applied to Whatman paper and chromatographed. BAT from control rats kept at 22 degrees C produced 79 +/- 30 pg T3/g protein/h. This value was significantly reduced in homogenates containing 1 or 5 mM zinc. In rats exposed to 4 degrees C, T3 production increased to 248 +/- 37 pg/mg protein/h. The addition of 100 microM, 1 or 5 mM zinc significantly decreased T3 production. The inhibitory action of zinc on BAT T4 deiodination may have a deleterious effect on BAT thermogenesis.
Subject(s)
Adipose Tissue, Brown/drug effects , Cadmium Chloride/toxicity , Cold Temperature , Mitochondria/drug effects , Adipose Tissue, Brown/physiology , Animals , Body Temperature Regulation/drug effects , Male , Mitochondria/metabolism , Oxygen Consumption , Rats , Rats, Wistar , Triiodothyronine/administration & dosageABSTRACT
Immunocytochemical localization of tryptamine in rat brain was performed at light and electron microscope level. The immunoreactive cells were present in the mesencephalon, pons and medulla, around the interpendicular nucleus in the reticular substantia nigra and in the giganto cellularis ventralis nucleus. Immunoreactive fibres were present in the dorsal region of the brain stem, and innervated the hippocampus, the ependymal epithelium and the corpus striatum. An ultrastructural study revealed that tryptamine was present only in neuronal perikaryon and processes.