Characteristics of intracellular proteolysis in the cells of Bacillus subtilis

The rate of total protein degradation down to acid-soluble products in the B. subtilis cells growing on a minimal medium is about 4--5% per hour. Under amino acid deficiency the rate of proteolysis depends on the allelic state of the relA gene, so that in the rel+ cells it increases two-fold, while in the rel- cells it remains low. Elimination of NH4+, PO43- and Mg2+ from the culture medium or an addition of NaN3 (8 mM) or 2,4-dinitrophenol (2 mM) results in 1.5--2.0-fold stimulation of proteolysis independently of the relA gene. In all cases studied the rate of proteolysis decreases after addition of chloramphenicol (100 micrograms/ml). It is proposed that chloramphenicol decreases the intracellular concentration of ppGpp, which is believed to exert pleiotropic alterations of cellular metabolism under condition of growth limitation. Quite different is the case of degradation of anomalous proteins synthesized in the presence of the lysine analog--S-2-aminoethylcystein. Degradation of anomalous proteins proceeds very rapidly (about 70% per hour); chloramphenicol (100 micrograms/ml) decreases the rate of proteolysis only two-fold. It was found that tetracycline (100 micrograms/ml) effectively inhibits the degradation of anomalous proteins. This activity of tetracycline was not observed in the presence of 50 mM of Mg2+ and seems to be dependent on the capacity of the antibiotic to form complexes with bivalent cations. These results reveal common features of control of proteolysis in the cells of B. subtilis and E. coli.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

The influence of chloramphenicol on synthesis of ribosomes and beta and beta' subunits of RNA polymerase]

The effect of low chloramphenicol concentrations on the biosynthesis of RNA, ribosomal proteins and RNA polymerase in E. coli CP 78 cells was studied. When protein synthesis was decreased by 50--70%, 14C-uracil incorporation in DNA increased twice, the rRNA synthesis being stimulated preferentially. In the presence of antibiotic the RNA/DNA ratio increased from 5,7 to 13,3. The differential rate of r-protein synthesis increased simultaneously with the stimulation of rRNA synthesis, so that alphar rises from 0,083 (without antibiotic) to 0,122 and 0,161 at 5 and 10 microgram/ml of chloramphenicol, respectively. The inhibition of protein synthesis by chloramphenicol is accompanied also by the increase of differential rate of synthesis of beta and beta' subunits of RNA polymerase. In the presence of 5 and 10 microgram/ml of chloramphenicol, alphap increased from 0,90% to 1,44 and 1,57%, respectively. It is assumed that the genes for beta and beta' subunits of RNA polymerase as the ribosomal genes are negatively controlled by guanosine tetraphosphate which intracellular concentration decreased in the presence of chloramphenicol. The known data on the influence of streptolydigin and rifampicin on the RNA polymerase biosynthesis are discussed in view of proposed hypothesis.     

Translation of poly U by ribosomes from rel+ and rel- E. coli strains]

A translation of polyU in a cell-free system from CP78 (relA+) and CP79 (relA-) E. coli strains is investigated. The strains studied no not differ in misreading at Mg2+ concentration of 20 mM and concentrations of 16 mM (optimal for phenylalanine incorporation) and 18-22 mM (optimal for leucine incorporation) respectively. It is found that leucine incorporation increases similarly in both strains under conditions simulating an amino acid deficience (in phenylalanine-free incubation medium): the ratio leucine(-phenylalanine)/leucine (+phenylalanine) is 3.5-4 at a concentration of enzymatic fraction protein being 15-30 mkg per example, and it is 2 st a concentration of enzymatic fraction of 60-180 mkg of protein. It is suggested that differences in activities of a number of enzymes under amino acid starvation in Rel+ and Rel- cells are not due to differences in the precision of mRNA translation, but depend on the activity of respective genes.     

Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.