Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.     

Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3

Atg4C/autophagin-3 is a member of a family of cysteine proteinases proposed to be involved in the processing and delipidation of the mammalian orthologues of yeast Atg8, an essential component of an ubiquitin-like modification system required for execution of autophagy. To date, the in vivo role of the different members of this family of proteinases remains unclear. To gain further insights into the functional relevance of Atg4 orthologues, we have generated mutant mice deficient in Atg4C/autophagin-3. These mice are viable and fertile and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, Atg4C-/--starved mice show a decreased autophagic activity in the diaphragm as assessed by immunoblotting studies and by fluorescence microscopic analysis of samples from Atg4C-/- GFP-LC3 transgenic mice. In addition, animals deficient in Atg4C show an increased susceptibility to develop fibrosarcomas induced by chemical carcinogens. Based on these results, we propose that Atg4C is not essential for autophagy development under normal conditions but is required for a proper autophagic response under stressful conditions such as prolonged starvation. We also propose that this enzyme could play an in vivo role in events associated with tumor progression.     

Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation

Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.     

Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium. This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene. Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins. Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene. In some combinations, negative complementation was observed. For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes. Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein. These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.     

Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5(-/-) mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Lysophosphatidic acid receptor-2 (LPA2)-mediated signaling enhances chemoresistance in melanoma cells treated with anticancer drugs

Molecular and Cellular Biochemistry - Parkinson’s disease (PD) is the second common age-related neurodegenerative disease. It is characterized by control loss of voluntary movements control,...     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

Phospholipid composition of rat megakaryocytes and its rearrangement in platelets

Rat platelets and their megakaryocyte precursors were examined for phospholipid composition. (1) The phospholipid composition of rat megakaryocytes, which were enriched and prepared from bone marrow cells, was almost identical to that of platelets. (2) The subclass composition of choline-containing glycerophospholipids (CGP) of rat megakaryocytes differed significantly from that of platelets: 1-alkenyl-2-acyl glycerophosphocholine (GPC) in megakaryocytes accounted for 29% of the total, whereas that in platelets was only 7%. (3) Rat platelets contained a larger amount of arachidonic acid than megakaryocytes, especially in ethanolamine-containing glycerophospholipids (EGP). (4) [32P]Phosphoric acid was significantly incorporated into megakaryocytes, whereas platelets showed little incorporation. On the other hand, the uptake of [3H]arachidonic acid into platelet phospholipids was about 15-times higher than that observed with megakaryocytes. (5) As reported previously for other blood cells, such as neutrophils and macrophages, the radioactivity of labeled arachidonic acid incorporated into CGP of platelets decreased, whereas that incorporated into EGP increased during a subsequent chase period. Hardly any such change was observed with megakaryocytes. These results suggest that the phospholipid composition of rat platelets is mainly determined at the time of thrombopoiesis, whereas the composition of molecular species is remodeled during circulation after thrombopoiesis.