The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny

Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.     

Utilization of alanine for glucose formation in isolated rabbit kidney-cortex tubules

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5-10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U-14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U-14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains.

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

In vitro transport of ornithine carbamoyltransferase precursor into rat liver mitochondria using a more homologous medium

The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.