Isolation of cis-3-Amino-l-Proline from Cultured Mycelia of Morchella esculenta Fr

cis-3-Amino-l-proline, identified once as a nonprotein amino acid from the fruiting bodies of Morchella esculenta Fr., was isolated also from the growth medium and cultured mycelia of the same fungus.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Hyperlipidemia in mast cell-deficient W/WV mice

Approximately 70% of the W/WV mice lacking mast cells due to a genetic defect showed hypertriglyceridemia combined with hypercholesterolemia. Increases of various magnitudes in chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein were observed in the plasma of W/WV mice compared to those in the plasma of congenic normal mice. The increase in these lipoproteins was seen even in normolipidemic W/WV mice. Activities of both lipoprotein lipase and hepatic triacylglycerol lipase in the plasma after heparin injection were markedly lower in the W/WV mice than in the congenic normal mice, although activities of both lipoprotein lipase in the heart and adipose tissue and hepatic triacylglycerol lipase in the liver were not decreased. These results suggest that the W/WV mice have genetic defects in one or more of the following: secretion of both lipases from their synthesising cells, transport to the endothelium, and anchoring to the endothelial surface. Heparin deficiency in these mice may be responsible for the impairment and, thereby, may partially contribute to the hyperlipidemia.     

Lipoxygenase, Hydroperoxide Lyase and Volatile C6-Aldehyde Formation from C18-Fatty Acids during Development of Phaseolus vulgaris L.

Kidney bean plants (Phaseolus vulgaris) were found to have thecapability to produce C₆-aldehydes (hexanal and hexenals) fromlinoleic and linolenic acids. The various organs tested hadlipoxygenase and hydroperoxide lyase activities responsiblefor the C₆-aldehyde formation. Young leaves showed relativelyhigh activities for C₆-aldehyde formation. However, the activitiesof the leaves decreased gradually with leaf expansion. Seedlingsand seeds containing cotyledons showed low activities for C₆-aldehydeformation, because of the occurrence of an inhibitory factorin the cotyledons. The substrate specificity of the enzymeswas essentially the same among the various developmental stagesof leaves tested. (Received February 5, 1982; Accepted March 19, 1982)     

Characteristic morphology of intracellular microcolonies of Legionella oakridgensis OR-10

Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.     

Discovery of the rpl10 Gene in Diverse Plant Mitochondrial Genomes and Its Probable Replacement by the Nuclear Gene for Chloroplast RPL10 in Two Lineages of Angiosperms

Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots.     

Phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) by Proline-Directed Protein Kinases and Its Dephosphorylation

Abstract: Excitatory amino acid (EAA) neurotransmitters may play a role in the pathophysiology of traumatic injury to the CNS. Although NMDA receptor antagonists have been reported to have therapeutic efficacy in animal models of brain injury, these compounds may have unacceptable toxicity for clinical use. One alternative approach is to inhibit the release of EAAs following traumatic injury. The present study examined the effects of administration of a novel sodium channel blocker and EAA release inhibitor, BW1003C87, or the NMDA receptor-associated ion channel blocker magnesium chloride on cerebral edema formation following experimental brain injury in the rat. Animals (n = 33) were subjected to fluid percussion brain injury of moderate severity (2.3 atm) over the left parietal cortex. Fifteen minutes after injury, the animals received a constant infusion of BW1003C87 (10 mg/kg, i.v.), magnesium chloride (300 µmol/kg, i.v.), or saline over 15 min (2.75 ml/kg/15 min). In all animals, regional tissue water content in brain was assessed at 48 h after injury, using the wet weight/dry weight technique. In saline-treated control animals, fluid percussion brain injury produced significant regional brain edema in injured left parietal cortex ( p < 0.001), the cortical area adjacent to the site of maximal injury ( p < 0.001), left hippocampus ( p < 0.001), and left thalamus ( p = 0.02) at 48 h after brain injury. Administration of BW1003C87 15 min postinjury significantly reduced focal brain edema in the cortical area adjacent to the site of maximal injury ( p < 0.02) and left hippocampus ( p < 0.01), whereas magnesium chloride attenuated edema in left hippocampus ( p = 0.02). These results suggest that excitatory neurotransmission may play an important role in the pathogenesis of posttraumatic brain edema and that pre- or post-synaptic blockade of glutamate receptor systems may attenuate part of the deleterious sequelae of traumatic brain injury.     

Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270) was obtained using sequence-based screening. From {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.Prolyl aminopeptidase (PAP) (EC 3.4.11.5), belonging to the S33 family, is an exopeptidase that catalyzes the hydrolysis of the N terminus prolyl residue of peptides or proteins. This family has catalytic Ser. To date, few applications of this enzyme for peptide synthesis have been reported. However, from the perspective of biotechnology, PAP might be a good tool for synthesizing proline-containing peptides by catalyzing aminolysis.Recently nutraceutical properties of peptides containing proline have received increasing attention. For example, prolyl hydroxyproline (Pro-Hyp) stimulates the growth of fibroblasts from mouse skin (11). Pro-Arg can protect against oxidative stress/damage and H₂O₂-induced human diploid fibroblast cell death (13). Furthermore, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro exhibit angiotensin I-converting enzyme-inhibiting activity (9). In addition to these dipeptides and tripeptides, a cyclic dipeptide (namely, diketopiperazine) containing proline shows several physiological functions. Cyclo[Pro-Pro] (cPP) exerts antibacterial activity against Micrococcus luteus and Pseudomonas aeruginosa (8). Caspase-3 activation by cyclo[Pro-Phe] in HT-29 cells has been described (3). However, its synthesis method has not been established. Enzymatic peptide synthesis presents a useful and desirable strategy because it can conduct specific reactions under milder conditions than those of chemical synthesis.Engineered endoserine proteases that have Cys substituted for catalytic Ser have also been applied for peptide synthesis since subtiligase was constructed by Abrahmsén et al. (1). Because of the weakened hydrolytic activity of the parent enzyme, it is considered that Ser/Cys-substituted protease can trap the substrate (acyl donor). Then, a nucleophilic reaction occurs between another substrate (acyl acceptor) and the trapped acyl donor (2). This is a so-called “aminolysis” reaction. Although aminolysis can conduct peptide synthesis in an aqueous solution, the problem of the necessity of using an N-protected amino acid as an acyl donor remains when using endoproteases.These problems would be solved using exoprotease as a catalyst, because N-terminal free amino groups of acyl donors are recognized by enzymes. It is rarely reported that exoprotease was applied for peptide synthesis, except in the report of Oshiro et al., in which Pro-Phe, Pro-Tyr, and Pro-Trp were synthesized (10). Recently our group reported that the Ser/Cys variant of exoprotease, aminolysin-S, has been constructed and has produced l-Phe-l-Phe ethyl ester and their derivatives from non-N-protected phenylalanine and phenylalanine ethyl ester as acyl donors in aqueous solution (12). However, aminolysin-S cannot produce proline-containing dipeptides.In this study, we describe a PAP from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270). Furthermore, synthesis of various proline peptides was attempted through catalysis by its Ser/Cys variant (scPAP14270) from proline ester and several amino acids and their esters in aqueous solution. A basic characterization to determine the effect of pH and the amount of substrate was conducted. Moreover, correlation was found between proline peptide synthesis and the log D value, which is the distribution coefficient between octanol and water, of acyl acceptors in aminolysis mediated by scPAP14270.