Effects of polyamines on the activities of Escherichia coli ribonuclease I and II.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.     

Studies on the restoration of the activities of Ribonucleases by polyamines in the presence of various ribonuclease inhibitors.

The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor.     

LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome

The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494. The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results suggest that the gene expression of the operon is repressed strongly under standard conditions.     

Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells.

We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB.     

Primary structure of the alpha-subunit of vacuolar-type Na(+)-ATPase in Enterococcus hirae. Amplification of a 1000-bp fragment by polymerase chain reaction.

A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by the polymerase chain reaction method, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na(+)-ATPase alpha-subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of Na(+)-ATPase alpha-subunit is highly homologous to the corresponding sequences of large (alpha) subunits of vacuolar (archaebacterial) type H(+)-ATPases, supporting our proposal [Kakinuma, Y. and Igarashi, K. (1990) FEBS Lett. 271, 97-101] that the Na(+)-ATPase of this organism belongs to the vacuolar-type ATPase.     

Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone.

It has been reported that Escherichia coli is able to grow in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) when ATP is produced by glycolysis (N. Kinoshita et al., J. Bacteriol. 160:1074-1077, 1984). We investigated the effect of CCCP on the osmotic adaptation of E. coli growing with glucose. When E. coli growing in rich medium containing CCCP was transferred to medium containing sucrose, its growth stopped for a while and then started again. This lag time was negligible in the absence of CCCP. The same results were obtained when the osmolarity was increased by N-methylglucamine-maleic acid. In addition to adapting itself to the hyperosmotic rich medium, E. coli adapted itself to hyperosmolarity in a minimal medium containing CCCP, again with a lag time. Hyperosmotic shock decreased the internal level of potassium ion rather than causing the accumulation of external potassium ion in the presence of CCCP. The internal amount of glutamic acid increased in cells growing in hyperosmotic medium in the presence and absence of CCCP. Large elevations in levels of other amino acids were not observed in the cells adapted to hyperosmolarity. Trehalose was detected only in hyperosmosis-stressed cells in the presence and absence of CCCP. These results suggest that E. coli can adapt to changes in the environmental osmolarity with a negligible accumulation of osmolytes from the external milieu but that the accumulation may promote the adaptation.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sj?gren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.