Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.     

Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.     

Longitudinal patterns of dissolved organic carbon concentration and suspended bacterial density along a blackwater river

Dissolved organic carbon (DOC) is the dominant form of carbon in transport in blackwater rivers, and bacteria are the major biological agents of its utilization. This study describes longitudinal patterns in DOC concentration and relates them to suspended bacterial populations in the channel. Concentrations of total DOC, three molecular weight fractions, and bacterial numbers were determined at 12 sites along the Ogeechee River in 1985–1986 and 1989 during periods of low and high discharge. Suspended bacterial populations were compared with DOC concentrations to determine if differences in bacterial abundance were related to longitudinal patterns of DOC concentration. Three distinct longitudinal patterns were observed: (1) The longitudinal pattern followed by both total and intermediate molecular weight DOC concentrations was a linear function of the geographic distance along the river. (2) During low flow conditions, there was a high degree of correspondence between patterns of bacterial numbers and low MW DOC (< 1000 apparent MW). (3) During periods of high discharge, the proportion of high (> 10,000) and intermediate (1000–10,000) MW fractions increased, and there was no longer a clear relationship between bacterial cells and low MW DOC.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.     

Isolation and re-association of the subunits from the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas

The component subunits of the pro-(carboxypeptidase A)–pro-(proteinase E) binary complex from pig pancreas were separated with a high recovery (80–95%) of their original potential activity. The isolated subunits and the reconstituted complex have properties similar to those of the corresponding natural species. The tryptic activation course of the pro-(carboxypeptidase A) subunit is substantially modified when bound to pro-(proteinase E), whereas the activation of pro-(proteinase E) is not dependent on this association.     

Independent temporal expression of two N-substituted aminoacyl-tRNA hydrolases during the development of Artemia salina.

Encysted embryos of the crustacean Artemia salina contain an enzymatic activity which hydrolyzes N-acetylphenylalanyl-tRNA to N-acetylphenylalanine and tRNA. The enzyme apparently does not hydrolyze other free or N-substituted aminoacyl-tRNAs. The levels of this enzyme do not significantly change during embryonic and early larval development. In contrast, an unspecific hydrolase active on several N-substituted aminoacyl-tRNAs is practically absent in the encysted embryos and during embryogenesis and appears abruptly during larval development. The independent temporal expression of these two hydrolases during Artemia salina differentiation makes this organism siuitable for the study of the physiological role of these enzymes.