Differential stain for mycobacteria that does not require heat.

A stain of 0.1% toluidine blue in a 1.0% aqueous solution of triethylene glycol followed by decolorization with acid-alcohol resulted in mycobacteria retaining the stain (violet), whereas non-acid-fast bacteria were decolorized.     

Iron-responsive degradation of iron-regulatory protein 1 does not require the Fe-S cluster

The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron-regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNA-binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.     

Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis.

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing beta(2)-adrenergic receptors (beta(2)ARs), sustained agonist treatment caused a time-dependant decrease in both beta(2)AR binding sites and immuno-detectable receptor. Blocking beta(2)AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasome-associated, were ineffective in preventing beta(2)AR down-regulation. Further evidence for an endocytosis-independent pathway of beta(2)AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous beta(2)AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block beta(2)AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for beta(2)AR down-regulation and that the inactivation of beta(2)ARs, leading to a reduction in binding sites, may occur at the plasma membrane.     

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.     

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.     

Evidence that incorporation of exogenous fatty acids into the phospholipids of Escherichia coli does not require acyl carrier protein.

Cells of an Escherichia coli acpS mutant were prepared with decreased intracellular concentrations (to 10% of the normal level) of the holo form of acyl carrier protein. These cells incorporated exogenous oleic acid into phospholipid at a normal rate.     

Miniplasmids of bacteriophage P1. I. Stringent plasmid replication does not require elements that regulate the lytic cycle

Defective prophages of bacteriophage P1Cm that lack about a third of the P1 genome were isolated. The known elements involved in the control of the lytic cycle are eliminated by deletion (c4, ant) or can be rendered non-functional by thermal denaturation (c1-ts) without killing the lysogen. Precise control over plasmid replication and segregation is retained in the absence of these controls on the lytic cycle. It is proposed that the plasmid and lytic cycle modes of P1 replication are regulated by separate mechanisms.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

Insulin-induced p21ras activation does not require protein kinase C, but a protein sensitive to phenylarsine oxide

Insulin treatment of fibroblasts overexpressing the insulin receptor causes a rapid accumulation of the GTP-bound form of p21ras. We have studied the involvement of protein kinase C (PKC) in, and the effect of phenylarsine oxide (PAO), a putative inhibitor of tyrosine phosphatase activity on, this process. Activation of p21ras was not observed when the cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and pretreatment with TPA for 16 h, sufficient to down-regulate PKC activity, did not abolish p21ras activation by insulin. These results show that PKC is not involved in the insulin-induced activation of p21ras. Pretreatment of the cells with PAO for 5 min completely blocked insulin-induced p21ras activation. Addition of 2,3-dimercaptopropanol prevented this inhibition by PAO. Also, addition of PAO after insulin stimulation could reverse the activation of p21ras. Since PAO did not affect overall phosphorylation of the insulin receptor beta-chain, we conclude that a PAO-sensitive protein is involved in the induction of p21ras activation by insulin.     

Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF(SLY1) E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.