Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Effects of Aging and Idiopathic Parkinson’s Disease on Tactile Temporal Order Judgment

It is generally accepted that the basal ganglia play an important role in interval timing that requires the measurement of temporal durations. By contrast, it remains controversial whether the basal ganglia play an essential role in temporal order judgment (TOJ) of successive stimuli, a behavior that does not necessarily require the measurement of durations in time. To address this issue, we compared the effects of idiopathic Parkinson’s disease (PD) on the TOJ of two successive taps delivered to each hand, with the arms uncrossed in one condition and crossed in another. In addition to age-matched elderly participants without PD (non-PD), we examined young healthy participants so that the effect of aging could serve as a control for evaluating the effects of PD. There was no significant difference between PD and non-PD participants in any parameter of TOJ under either arm posture, although reaction time was significantly longer in PD compared with non-PD participants. By contrast, the effect of aging was apparent in both conditions. With their arms uncrossed, the temporal resolution (the interstimulus interval that yielded 84% correct responses) in elderly participants was significantly worse compared with young participants. With their arms crossed, elderly participants made more errors at longer intervals (~1 s) than young participants, although both age groups showed similar judgment reversal at moderately short intervals (~200 ms). These results indicate that the basal ganglia and dopaminergic systems do not play essential roles in tactile TOJ involving both hands and that the effect of aging on TOJ is mostly independent of the dopaminergic systems.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

Complete Genome Sequence of Finegoldia magna, an Anaerobic Opportunistic Pathogen

Finegoldia magna (formerly Peptostreptococcus magnus), a memberof the Gram-positive anaerobic cocci (GPAC), is a commensalbacterium colonizing human skin and mucous membranes. Moreover,it is also recognized as an opportunistic pathogen responsiblefor various infectious diseases. Here, we report the completegenome sequence of F. magna ATCC 29328. The genome consistsof a 1 797 577 bp circular chromosome and an 189 163bp plasmid (pPEP1). The metabolic maps constructed based onthe genome information confirmed that most F. magna strainscannot ferment most sugars, except fructose, and have variousaminopeptidase activities. Three homologs of albumin-bindingprotein, a known virulence factor useful for antiphagocytosis,are encoded on the chromosome, and one albumin-binding proteinhomolog is encoded on the plasmid. A unique feature of the genomeis that F. magna encodes many sortase genes, of which substratesmay be involved in bacterial pathogenesis, such as antiphagocytosisand adherence to the host cell. The plasmid pPEP1 encodes sevensortase and seven substrate genes, whereas the chromosome encodesfour sortase and 19 substrate genes. These plasmid-encoded sortasesmay play important roles in the pathogenesis of F. magna byenriching the variety of cell wall anchored surface proteins.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.