Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Dopaminergic modulation of salt sensitivity in patients with essential hypertension

The present study was designed to investigate the possible role of dopaminergic mechanisms in contributing to the pathogenesis of hypertension in salt sensitive patients. Eighteen patients with essential hypertension were studied while under a diet ranging from low salt to high salt, which enabled a classification in "salt-sensitive" (SS) and "nonsalt-sensitive" (NSS) groups based on a tentative criteria of a 10% increase of mean blood pressure with high salt diet. The SS patients showed reduced urinary excretion of sodium as compared with that from NSS patients. Urinary norepinephrine excretion in all patients with salt loading was suppressed, but urinary excretion of epinephrine showed a tendency to increase in SS patients after salt loading. Urinary excretion of dopamine increased in NSS patients with salt loading, but did not change in SS patients. To further evaluate the role of dopaminergic mechanisms in salt-sensitive hypertension, metoclopramide, a dopamine antagonist, was injected intravenously to all patients. With salt loading, plasma aldosterone levels increased after injection of metoclopramide in NSS patients, but did not change in SS patients. These results suggest that salt-sensitive hypertension is modulated by dopaminergic activity, which in turn is attenuated in SS patients. Decreased dopaminergic activity induced sodium retention both by a direct effect on the kidney as well as indirectly via relatively increased aldosterone secretion. Both mechanisms would help to increase intravascular volume and blood pressure in salt-sensitive hypertension.     

Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.     

Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5'-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5'-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47?- W-383) protein was autopolymerized to form filamentous structures of about 80?nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.     

Relationship between amylase and fluid secretion in the isolated perfused whole parotid gland of the rat

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly at 40-120 microliter/g-min (average plateau was 60 microliter/g-min), whereas amylase secretion exhibited an initial peak (10 mg maltose/30 s per g wet w. of the gland), followed by a rapid decrease to reach a plateau level of 1 mg maltose/30 s later than 1.5-2 min. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion to 15 mg maltose/30 s accompanied by the increase in oxygen consumption. However, the fluid secretion exhibited a rather gradual decrease. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed -exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. During washout of secretagogues, lysosomal digestion of excess membrane took place.     

Roles of the extreme N‐terminal region of FliH for efficient localization of the FliH–FliI complex to the bacterial flagellar type III export apparatus

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self‐assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH–FliI complex also binds to the C ring of the basal body through a FliH–FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI–YFP by fluorescence microscopy. FliI–YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI–YFP formed a complex with FliHΔ1 (missing residues 2–10) but the complex did not show any localization. FliHΔ1 did not interact with FliN, and alanine‐scanning mutagenesis revealed that only Trp‐7 and Trp‐10 of FliH are essential for the interaction with FliN. Overproduction of the FliH–FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)‐FliI nor FliH(W10A)‐FliI complexes did, suggesting that Trp‐7 and Trp‐10 of FliH are also required for efficient localization of the FliH–FliI complex to the export gate.     

Potential impacts of flooding events and stream modification on an endangered endemic plant, <Emphasis Type="Italic">Schoenoplectus gemmifer</Emphasis> (Cyperaceae)

River management often conflicts with the conservation of species in river ecosystems. In Japan, almost all river systems have been covered by concrete walls. Such river improvement works caused critical damage to river ecosystems. Here we report the ongoing extinction process of a rare aquatic plant by several consecutive heavy rains. The aquatic plant, Schoenoplectus gemmifer C. Sato, T. Maeda & Uchino, is an extremely rare endemic species that is strictly associated with springwater. The species is only found in 23 locations in Japan, including only two major habitats: Hamamatsu and Oita. We monitored the population fluctuations of S. gemmifer at three river systems in Hamamatsu. In the largest habitat, Higashikanda River, the population size of the species decreased to nearly 1/10th in 2004, due to several severe floods. Spatial and temporal records exhibit four stages of damaging process. The stepwise damages were found to be caused by a rapid flow of water accelerated by the river improvement work (made in 1985). The reproduction and growth by seeds and gemmae did not evidently cover the losses by flood washed out. In the other two rivers, one was extinct and the other is now at the risk of extinction. The modified river structures may be responsible for the near-extinction of S. gemmifer in Hamamatsu area. We propose two policies for the conservation of this species: (1) the artificial cultivation of gemmae and seedlings and (2) the modification of river structure to decrease the number of washed-out plants. In particular, it is important to decrease the water velocity at floods by some methods.     

HIF-1α is necessary to support gluconeogenesis during liver regeneration

Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1α (HIF-1α) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1α in the regulation of gluconeogenesis under liver regeneration.     

Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1

A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.