A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.     

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): deciphering the basis for nucleotide selection

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N6-methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2'-O-methyladenosine, 2'-deoxy-2'-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3'-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.     

The protein kinase C inhibitor, calphostin C, inhibits succinate-dependent mitochondrial reduction of MTT by a mechanism that does not involve protein kinase C.

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.     

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.     

Mutant alpha-latrotoxin (LTXN4C) does not form pores and causes secretion by receptor stimulation: this action does not require neurexins

Alpha-latrotoxin (LTX) causes massive release of neurotransmitters via a complex mechanism involving (i) activation of receptor(s) and (ii) toxin insertion into the plasma membrane with (iii) subsequent pore formation. Using cryo-electron microscopy, electrophysiological and biochemical methods, we demonstrate here that the recently described toxin mutant (LTXN4C) is unable to insert into membranes and form pores due to its inability to assemble into tetramers. However, this mutant still binds to major LTX receptors (latrophilin and neurexin) and causes strong transmitter exocytosis in synaptosomes, hippocampal slice cultures, neuromuscular junctions, and chromaffin cells. In the absence of mutant incorporation into the membrane, receptor activation must be the only mechanism by which LTXN4C triggers exocytosis. An interesting feature of this receptor-mediated transmitter release is its dependence on extracellular Ca2+. Because Ca2+ is also strictly required for LTX interaction with neurexin, the latter might be the only receptor mediating the LTXN4C action. To test this hypothesis, we used conditions (substitution of Ca2+ in the medium with Sr2+) under which LTXN4C does not bind to any member of the neurexin family but still interacts with latrophilin. We show that, in all the systems tested, Sr2+ fully replaces Ca2+ in supporting the stimulatory effect of LTXN4C. These results indicate that LTXN4C can cause neurotransmitter release just by stimulating a receptor and that neurexins are not critical for this receptor-mediated action.     

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.     

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes.

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.     

Mouth development in the Senegal bichir Polypterus senegalus does not involve the oropharyngeal membrane: possible implications for the ecto‐endoderm boundary and tooth initiation

Polypterid fishes are considered the basal‐most group of extant actinopterygians and thus may serve as a direct link to understand the evolution of the first bony fishes. Embryonic and larval specimens, however, are extremely scarce, making it difficult to study their developmental patterns and processes. During the past few years we collected many embryos and larvae of the Senegal bichir Polypterus senegalus and in this paper we describe some novel observations concerning the supposed ecto‐endoderm border in the mouth and consecutive initiation of dental development. The mouth of the Senegal bichir does not develop via a classical oropharyngeal membrane but instead, opening the mouth occurs via a separation of the upper and lower jaws that are connected by epithelial bridges. These structures are bilaterally symmetrical and are found invariantly at places of the earliest tooth bud development. It is suggested that the epithelial bridges may represent the ecto‐endoderm bordering zone, are both structurally and functionally homologous to the oropharyngeal membrane and consequently it is hypothesized that the epithelial bridges and the developmental factors producing them play a key role in initiation and early distribution of the particular dental domains.     

Additional rabbit IgA allotypes, f69, f70, g76,g77: control by the C alphaf and C alphag loci.

Genetic and immunochemical studies have led to the identification of four additional rabbit IgA allotypes controlled by the Calphaf and Calphag loci. The folowing linkage combinations of the VHa and the 'new' alleles were observed among the populations of rabbits studied: a1f70g76, a1f69g77, and a2f69g77. Cross-reactions among g74, g76, and g77 molecules with various anti-g anti-allotype antisera indicate that the IgA-g allotypic specificities are comprised of multiple antigenic determinants. These studies provide a basis for further understanding of the evolution and gnetic control of the immunoglobulin heavy chain chromosomal region.     

c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.