Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB₁-C-A-B₂, from which two proteins, OdhAB₁C and OdhAB₂C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB₁ and OdhB₂ in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.     

Cloning and characterization of novel snake venom proteins that block smooth muscle contraction.

In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.     

Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

The Ca²⁺ release-activated Ca²⁺ channel is a principal regulator of intracellular Ca²⁺ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca²⁺ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca²⁺ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.Ca²⁺ is an intracellular second messenger that plays important roles in various physiological functions such as immune response, muscle contraction, neurotransmitter release, and cell proliferation. Intracellular Ca²⁺ is mainly stored in the endoplasmic reticulum (ER).² This ER system is distributed through the cytoplasm from around the nucleus to the cell periphery close to the plasma membrane. In non-excitable cells, the ER releases Ca²⁺ through the inositol 1,4,5-trisphosphate (IP₃) receptor channel in response to various signals, and the Ca²⁺ store is depleted. Depletion of Ca²⁺ then induces Ca²⁺ influx from outside the cell to help in refilling the Ca²⁺ stores and to continue Ca²⁺ rise for several minutes in the cytoplasm (1, 2). This Ca²⁺ influx was first proposed by Putney (3) and was named store-operated Ca²⁺ influx. In the immune system, store-operated Ca²⁺ influx is mainly mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) current, which is a highly Ca²⁺-selective inwardly rectified current with low conductance (4, 5). Pathologically, the loss of CRAC current in T cells causes severe combined immunodeficiency (6) where many Ca²⁺ signal-dependent gene expressions, including cytokines, are interrupted (7). Therefore, CRAC current is necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically characterized as essential components of the CRAC channel (8–12). They are separately located in the plasma membrane and in the ER membrane; co-expression of these proteins presents heterologous CRAC-like currents in various types of cells (10, 13–15). Both of them are shown to be expressed ubiquitously in various tissues (16–18). STIM1 senses Ca²⁺ depletion in the ER through its EF hand motif (19) and transmits a signal to Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory component for some transient receptor potential canonical channels (20, 21), it is believed from the mutation analyses to be the pore-forming subunit of the CRAC channel (8, 22–24). In the steady state, both Orai1 and STIM1 molecules are dispersed in each membrane. When store depletion occurs, STIM1 proteins gather into clusters to form puncta in the ER membrane near the plasma membrane (11, 19). These clusters then trigger the clustering of Orai1 in the plasma membrane sites opposite the puncta (25, 26), and CRAC channels are activated (27).Orai1 has two homologous genes, Orai2 and Orai3 (8). They form the Orai family and have in common the four transmembrane (TM) segments with relatively large N and C termini. These termini are demonstrated to be in the cytoplasm, because both N- and C-terminally introduced tags are immunologically detected only in the membrane-permeabilized cells (8, 9). The subunit stoichiometry of Orai1 is as yet controversial: it is believed to be an oligomer, presumably a dimer or tetramer even in the steady state (16, 28–30).Despite the accumulation of biochemical and electrophysiological data, structural information about Orai1 is limited due to difficulties in purification and crystallization. In this study, we have purified Orai1 in its tetrameric form and have reconstructed the three-dimensional structure from negatively stained electron microscopic (EM) images.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Higher serum bone alkaline phosphatase as a predictor of mortality in male hemodialysis patients

AimsHigher serum alkaline phosphatase predicts lower mortality in chronic kidney disease and hemodialysis patients without liver dysfunction because it reflects high bone turnover. The purpose of our study was to compare the significance of serum bone alkaline phosphatase (BAP) with that of other bone markers in prediction of all-cause mortality(ACM) in male hemodialysis patients.Main methodsThe study was performed for 5 years. Serum BAP, intact osteocalcin (iOC), ß-CrossLaps (CTX), and intact parathyroid hormone (iPTH) were measured in 196 male hemodialysis patients without radiographic fracture. Their day-to-day variation during 5 consecutive days and diurnal variation were determined in 13 healthy males.Key findingsThe patients were divided into higher and lower groups based on serum levels of bone markers(mean ± SD: iPTH 218.6 ± 214.5 pg/ml, BAP 23.6 ± 12.2U/L, iOC 42.8 ± 45.2 ng/ml, CTX 1.71 ± 1.23 nmol/L BCE). In Kaplan–Meier analysis, the higher BAP group had significantly higher ACM than the lower BAP group (P = 0.013), whereas mortality did not differ between the higher and lower groups in other markers. Cox regression hazard analysis identified higher log BAP as a significant independent predictor [hazard ratio(HR) 8.32(95%CI:1.18–58.98)] for ACM after adjustment for various factors including pre-existing cardiovasucular disease, presence of DM. The significant association of mortality with serum BAP alone, in contrast with other markers including CTX [HR0.64 (95%CI:0.16–2.47)], iOC [HR0.97(95%CI:0.36–2.64)], iPTH [HR0.84(95%CI:0.44–1.60)],it may be due to the narrower day-to-day variation and the absence of diurnal variation in serum BAP compared to other markers.SignificanceHigher serum BAP may be a predictor of ACM in male hemodialysis patients.     

Long-term feeding on powdered food causes hyperglycemia and signs of systemic illness in mice

Aims

Dietary habits are crucial factors affecting metabolic homeostasis. However, few animal experiments have addressed the effects of long-term feeding with soft food on parameters reflecting systemic health.

Main methods

Using mice, we compared the effects of short (3 days) and long (17 weeks from weaning) feeding periods between powdered food and normal pellet food on the levels of blood glucose, serum levels of insulin, catecholamines, and corticosterone, blood pressure, and/or social interaction behaviors. In addition, the effects of a human glucagon-like peptide-1 analog, liraglutide (a new drug with protective effects against neuronal and cardiovascular diseases), were compared between the powder and pellet groups.

Key finding

(i) Powdered food, even for such a short period, resulted in a greater glycemic response than pellet food, consistent with powdered food being more easily digested and absorbed. (ii) Long-term feeding on powdered food induced hyperglycemia and related systemic signs of illness, including increases in serum adrenaline, noradrenaline, and corticosterone, higher blood pressures (especially diastolic), and increased social interaction behaviors. (iii) Liraglutide, when administered subcutaneously for the last 2 weeks of the 17-week period of feeding, improved these changes (including those in social interaction behaviors).

Significance

The hyperglycemia associated with long-term powdered-food feeding may lead to certain systemic illness signs, such as elevations of blood glucose, hypertension, and abnormal behaviors in mice. Mastication of food of adequate hardness may be very important for the maintenance of systemic (physical and mental) health, possibly via reduction in the levels of blood glucose and/or adrenal stress hormones (catecholamines and glucocorticoids).