Quantification of polyprenyl diphosphates in Escherichia coli cells using high-performance liquid chromatography.

Dephosphorylation of undecaprenyl diphosphate is a crucial step in the synthesis of undecaprenyl phosphate, which is essential for cell wall synthesis. We have developed a method for the quantification of intracellular polyprenyl diphosphates, which have never before been measured directly. Polyprenyl phosphates and diphosphates prepared by chemical phosphorylation of polyprenols from Staphylococcus aureus were used to establish the conditions for fractionation by ion-exchange chromatography and high-performance liquid chromatography (HPLC). By using an elution solvent containing tetraethylammonium phosphate as an ion-pair reagent for HPLC, polyprenyl phosphate and polyprenyl diphosphate with carbon numbers from 40 to 55 could be detected as separate peaks from the reversed-phase column. This analytical method was applied to lipids extracted from Escherichia coli to determine the intracellular levels of octaprenyl phosphate, undecaprenyl phosphate, octaprenyl diphosphate, and undecaprenyl diphosphate. This is the first report of separate measurement of cellular levels of polyprenyl phosphates and polyprenyl diphosphates.

CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation.

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.

Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo.

Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca2+/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210.

Autoactivation of C-terminally truncated Ca2+/calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation.

Ca2+/calmodulin-dependent protein kinase I isoforms (CaMKIα, ß, γ, and δ) play important roles in Ca2+ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser296, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser296, which might be a clue to understand the physiological regulation of CaMKIδ isoform.

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea.

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca2+/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca2+/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca2+-signaling in C. cinerea.

High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.

We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay.

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.

Caenibacillus caldisaponilyticus gen. nov., sp. nov., a thermophilic, spore-forming and phospholipid-degrading bacterium isolated from acidulocompost.

A thermophilic and phospholipid-degrading bacterium, designated strain B157T, was isolated from acidulocompost, a garbage compost processed under acidic conditions at moderately high temperature. The organism was Gram-stain-positive, aerobic, spore-forming and rod-shaped. Growth was observed to occur at 40-65 °C and pH 4.8-8.1 (optimum growth: 50-60 °C, pH 6.2). The strain was catalase- and oxidase-positive. The cell wall contained meso-diaminopimelic acid, alanine, glutamic acid and galactose. The predominant respiratory quinone was menaquinone-7 (MK-7) and the major fatty acids were anteiso-C17 : 0 and iso-C17 : 0. Comparative 16S rRNA gene sequence analysis showed that strain B157T was related most closely to Tuberibacillus calidus 607T (94.8 % identity), and the phylogenetic analysis revealed that it belonged to the family Sporolactobacillaceae. The DNA G+C content was determined as 51.8 mol%. In spite of many similarities with the type strains of members of the family Sporolactobacillaceae, genotypic analyses suggest that strain B157T represents a novel species of a new genus, Caenibacilluscaldisaponilyticus gen. nov., sp. nov. The type strain of Caenibacilluscaldisaponilyticus is B157T (=NBRC 111400T=DSM 101100T).

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.

(7S)-Kaitocephalin as a potent NMDA receptor selective ligand.

A structure-activity relationship (SAR) study of kaitocephalin (KCP), known to be a potent naturally occurring NMDA receptor ligand, was performed. This study led us to the discovery of (7S)-kaitocephalin as a highly selective NMDA receptor ligand. It displayed a 22-fold higher degree of selectivity for the NMDA receptor over KCP, though the binding affinity of (7S)-KCP [Ki = 29 nM] was 3.7-fold less potent than that of KCP [Ki = 7.8 nM].

Cytotoxicity of CdSe-based quantum dots incorporated in glass nanoparticles evaluated using human keratinocyte HaCaT cells.

CdSe quantum dots (QDs) are potential fluorescent reagents, but leakage of Cd and Se often induces cytotoxicity. Here we prepared CdSe-based QDs with glass to reduce their leakage and examined their cytotoxicity using keratinocyte cells. The cytotoxicity of the QDs with glass was obviously lower than that of the commercial QDs with polymer, suggesting their safety for biological applications.

2-Hydrazinoquinoline: a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry to detect gaseous carbonyl compounds.

The sensitivity, range of applications, and reaction mechanism of 2-hydrazinoquinoline as a reactive matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were examined. Using a reaction chamber (125L) equipped with a stirring fan and a window for moving the MALDI-MS plate and volatile samples in and out, the sensitivities of 2-hydrazinoquinoline to gaseous aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and n-butyraldehyde) and ketones (acetone, methyl ethyl ketone, and methyl isobutyl ketone) were determined to be at least parts per million (ppm) levels. On the other hand, carboxylic acids (formic acid, acetic acid, propionic acid, and butyric acid) and esters (ethyl acetate, pentyl acetate, isoamyl acetate, and methyl salicylate) could not be detected by 2-hydrazinoquinoline in MALDI-MS. In addition to 2,4-dinitrophenylhydrazine, a common derivatization reagent for analyzing carbonyl compounds quantitatively in gas chromatography and liquid chromatography, the dissolution of 2-hydrazinoquinoline in an acidic solution, such as trifluoroacetic acid, was essential for its function as a reactive matrix for MALDI- MS.

Dynamics of hydroxyoctadecadienoic acid in epilepsy patients treated with valproic acid.

BACKGROUND: The effects of valproic acid (VPA) on oxidative stress are controversial due to differences in experimental conditions. Recently, total hydroxyoctadecadienoic acid (tHODE), derived from linoleic acid, was proposed as a potent biomarker to evaluate oxidative stress. METHODS: The subjects consisted of 10 new-onset epilepsy patients treated with VPA. We measured plasma tHODE consecutively for 1 year to evaluate the degree of oxidative stress and then compared plasma tHODE at the beginning and the end of experiments with the peak level. Ten age-matched healthy subjects were recruited as a control group and their plasma tHODE was compared to the initial plasma tHODE of the epilepsy group. Measurements were done using liquid chromatography-tandem mass spectrometry. RESULTS: Mean initial plasma tHODE in the epilepsy group was 165.2 ± 76.8 nmol/L, which was not significantly different from that of the control group (199.3 ± 62.5 nmol/L). In five epilepsy patients, plasma tHODE increased above the pathological level in 6 months, but returned to normal within 1 year. In the whole group, the difference plasma tHODE between peak, at the beginning and 1 year later, was significant. CONCLUSION: Oxidative stress generated by VPA increased temporarily, but decreased to normal after 1 year. it is reasonable to be concerned about the effects of oxidative stress, especially at the start of VPA treatment.

The Phosphatase-Resistant Isoform of CaMKI, Ca²âº/Calmodulin-Dependent Protein Kinase Iδ (CaMKIδ), Remains in Its "Primed" Form without Ca²âº Stimulation.

Ca²âº/calmodulin-dependent protein kinase I (CaMKI) is known to play pivotal roles in Ca²âº signaling pathways. Four isoforms of CaMKI (α, ß, γ, and δ) have been reported so far. CaMKI is activated through phosphorylation by the upstream kinase, CaMK kinase (CaMKK), and phosphorylates downstream targets. When CaMKI was transiently expressed in 293T cells, CaMKIα was not phosphorylated at all under low-Ca²âº conditions in the cells. In contrast, we found that CaMKIδ was significantly phosphorylated and activated to phosphorylate cAMP response element-binding protein (CREB) under the same conditions. Herein, we report that the sustained activation of CaMKIδ is ascribed to its phosphatase resistance resulting from the structure of its N-terminal region. First, we examined whether CaMKIδ is more readily phosphorylated by CaMKK than CaMKIα, but no significant difference was observed. Next, to compare the phosphatase resistance between CaMKIα and CaMKIδ, we assessed the dephosphorylation of the phosphorylated CaMKIs by CaMK phosphatase (CaMKP/PPM1F). Surprisingly, CaMKIδ was hardly dephosphorylated by CaMKP, whereas CaMKIα was significantly dephosphorylated under the same conditions. To date, there have been no detailed reports concerning dephosphorylation of CaMKI. Through extensive analysis of CaMKP-catalyzed dephosphorylation of various chimeric and point mutants of CaMKIδ and CaMKIα, we identified the amino acid residues responsible for the phosphatase resistance of CaMKIδ (Pro-57, Lys-62, Ser-66, Ile-68, and Arg-76). These results also indicate that the phosphatase resistance of CaMKI is largely affected by only several amino acids in its N-terminal region. The phosphatase-resistant CaMKI isoform may play a physiological role under low-Ca²âº conditions in the cells.

Molecular characterization of the genes involved in the secretion and immunity of lactococcin Q, a two-peptide bacteriocin produced by Lactococcus lactis QU 4.

Lactococcin Q is a two-peptide (Qα and Qß) bacteriocin produced by Lactococcus lactis QU 4, which exhibits specific antimicrobial activity against L. lactis strains. The lactococcin Q gene cluster (approximately 4.5 kb) was sequenced and found to include genes encoding lactococcin Q immunity (laqC), an ATP-binding cassette transporter (laqD) and a transport accessory protein (laqE), downstream of the lactococcin Q structural genes (laqA and laqB). In addition, the gene cluster showed high sequence identity with that of a lactococcin Q homologue bacteriocin, lactococcin G. Heterologous expression studies showed that LaqD was responsible for lactococcin Q secretion in a manner dependent on LaqE expression, and that LaqC conferred self-immunity to lactococcin Q and cross-immunity to lactococcin G. Amino acid alignment of both lactococcin transporters revealed that LaqD contains an insertion (160-168 residues) that is essential for lactococcin Q secretion, as L. lactis cells that expressed LaqDΔ160-168 were devoid of this function. Additional experiments demonstrated that the LaqDΔ160-168 mutant was, however, able to secrete lactococcin G, suggesting that the insertion is necessary only for the lactococcin Q secretion by LaqD. This report demonstrates the biosynthetic mechanism of lactococcin Q/G-type bacteriocins and the complementarity of the genes responsible for the secretion of lactococcins Q and G.

Photothermic regulation of gene expression triggered by laser-induced carbon nanohorns.

The development of optical methods to control cellular functions is important for various biological applications. In particular, heat shock promoter-mediated gene expression systems by laser light are attractive targets for controlling cellular functions. However, previous approaches have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, which have poor penetration into biological tissue. Biological tissue is relatively transparent to light inside the diagnostic window at wavelengths of 650-1,100 nm. Here we present a unique optical biotechnological method using carbon nanohorn (CNH) that transforms energy from diagnostic window laser light to heat to control the expression of various genes. We report that with this method, laser irradiation within the diagnostic window resulted in effective heat generation and thus caused heat shock promoter-mediated gene expression. This study provides an important step forward in the development of light-manipulated gene expression technologies.

Extraordinary denaturant tolerance of keratinolytic protease complex assemblies produced by Meiothermus ruber H328.

A moderately thermophilic bacterial strain, Meiothermus ruber H328, can efficiently solubilize intact chicken feathers by aerobic cultivation at 55 °C for 6 days. The keratinolytic proteases extracellularly secreted by the strain were partially purified by an ultrafiltration system and a size-exclusion column chromatography, and thus were found to be two different sizes of macromolecules with an extremely high molecular mass like the sizes of virus and DNA (peak 1 fraction) and with a molecular mass of larger than 500 kDa (peak 2 fraction). They formed protein complex assemblies that were composed of multiple but different proteins. The peak 1 fraction showed more thermophilic characteristics than did the peak 2 fraction in temperature dependence and thermal stability. By contrast, they comparably showed extraordinary resistance to powerful denaturants, SDS at 30 % (w/v) and organic solvents (methanol, ethanol, acetonitrile, acetone, and chloroform) at 40 % (v/v) at 60 °C for 30 min. The extraordinary denaturant tolerance and the large molecular size of the keratinolytic protease complex assemblies suggest the possibility that those may be lipophilic and have the structure of partial membrane fractions, or membrane vesicles, which are exfoliated from the outer membrane of the cells.