Targeting Determinants and Proposed Evolutionary Basis for the Sec and the Delta pH Protein Transport Systems in Chloroplast Thylakoid Membranes

Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems. Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity. The results showed that: (a) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system. A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway. This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.     

Coloboma Hyperactive Mutant Mice Exhibit Regional and Transmitter-Specific Deficits in Neurotransmission

Abstract: The mouse mutant coloboma ( Cm /+), which exhibits profound spontaneous hyperactivity and bears a deletion mutation on chromosome 2, including the gene encoding synaptosomal protein SNAP-25, has been proposed to model aspects of attention-deficit hyperactivity disorder. Increasing evidence suggests a crucial role for SNAP-25 in the release of both classical neurotransmitters and neuropeptides. In the present study, we compared the release of specific neurotransmitters in vitro from synaptosomes and slices of selected brain regions from Cm /+ mice with that of +/+ mice. The release of dopamine (DA) and serotonin (5-HT) from striatum, and of arginine vasopressin and corticotropin-releasing factor from hypothalamus and amygdala is calcium-dependent. Glutamate release from and content in cortical synaptosomes of Cm /+ mice are greatly reduced, which might contribute to the learning deficits in these mutants. In dorsal striatum of Cm /+ mutants, but not ventral striatum, KCI-induced release of DA is completely blocked and that of 5-HT is significantly attenuated, suggesting that striatal DA and 5-HT deficiencies may be involved in hyperactivity. Further, although acetylcholine failed to induce hypothalamic corticotropin-releasing factor release from Cm /+ slices, restraint stress increased plasma corticosterone levels in Cm /+ mice to a significantly higher level than in +/+ mice, suggesting an important role for arginine vasopressin in hypothalamic-pituitary-adrenal axis activation. These results suggest that reduced SNAP-25 expression may contribute to a region-specific and neurotransmitter-specific deficiency in neurotransmitter release.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Abstract: The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 µ M bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 m M methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of d -[³H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d -[³H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d -[³H]aspartate were competing with labeled vesicles for exocytosis. As d -[³H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2-10 destaining and d -[³H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.     

Ethanol Specifically Inhibits NMDA Receptors with Affinity for Ifenprodil in the Low Micromolar Range in Cultured Cerebellar Granule Cells

Abstract: The effect of ethanol on the intracellular Ca²⁺ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCI concentrations has been studied using image analysis. The cells grown in low KCI displayed high sensitivity for glycine. The subtype-selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high-potency component in these cultures. In cells grown in high KCI the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.     

Matrix Metalloproteinases in Dog Brains Exhibiting Alzheimer-Like Characteristics

Abstract: We have previously reported that the amount of the neuronal matrix metalloproteinase (MMP) MMP-9, capable of cleaving β-amyloid_1–40 predominantly at Leu³⁴-Met³⁵, is increased in a latent form in hippocampal specimens from AD patients and have suggested that the lack of activation of this enzyme may contribute to the deposition of β-amyloid in plaques. The current study addresses whether similar matrix proteinases are detectable in amyloid-positive and -negative brain specimens of aged beagles. Using quantitative zymography, three major neutral proteinases with molecular masses of 60, 95, and 280 kDa were readily detected. These enzymes have the characteristics of MMPs because they were inhibited by EDTA and 1,10-phenanthroline, and their activities were restored by addition of both Ca²⁺ and Zn²⁺. The 95- and 280-kDa proteinases cross-reacted with specific monoclonal antibodies to human MMP-9 (gelatinase B; EC 3.4.24.35). Canine MMP-9 was latent because activation by organomercurial treatment resulted in a characteristic decrease in molecular mass. Statistical analysis revealed no difference in the 60-kDa proteinase activity in amyloid-positive and -negative brain specimens. However, significantly increased amounts of latent MMP-9 were observed in amyloid-positive brain specimens ( p ≤ 0.05) compared with amyloid-negative brain specimens. The observations document that changes in MMP-9 expression in amyloid-positive beagle brains are similar to those reported in the human Alzheimer's disease hippocampus and suggest the possibility that insufficient activation of MMP-9 may contribute to β-amyloid accumulation, a hypothesis that needs to be further investigated.     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSIS SIMILIS (CHLOROPHYTA):FLAGELLAR AND BASAL BODY DEVELOPMENTAL CYCLE1

Flagellar and basal body development during cell division was studied in the biflagellate green alga Spermatozopsis similis Preisig et Melkonian by light microscopy of immobilized living cells, statistical analysis of flagellar lengths during the cell cycle, and electron microscopy of cells and isolated cytoskeletons. Interphase cells display two flagella of unequal/subequal length. An eyespot located in an anterior lobe of the chloroplast is connected to the basal body bearing the shorter flagellum by means of a five-stranded microtubular root. Until cell division, the two parental flagella attain the same length. During cell division, each cell forms two new flagella that grow to a length of 1.5 μm before they are distributed in a semiconservative fashion together with the parental flagella to the two progeny cells at cytokinesis. During the following interphase, the flagella newly formed during the preceding cell division grow to attain the same length as the parental flagella until the subsequent cell division. The shorter of the two flagella of a cell thus represents the developmentally younger flagellum, which transforms to the mature state during two consecutive cell cycles. Interphase cells display only two flagella-bearing basal bodies; two nascent basal bodies are formed during cell division and are connected to the microtubular d-roots of respective parental basal bodies with which the newly formed basal bodies are later distributed to the progeny cells. During segregation, basal body pairs shaft into the 11/5 o'clock direction, thus conserving the 1/7 o'clock configuration of basal body pairs of interphase cells. Prior to chloroplast and cell division, an eyespot is newly formed near the cell posterior in close association with a 1s microtubular root, while the parental eyespot is retained. During basal body segregation, eyespot-root connections for both the old and newly formed eyespots are presumably lost, and new associations of the eyespots with the 2s roots of the newly formed basal bodies are established during cytokinesis. The significance of this “eyespot-flagellar root developmental cycle” for the absolute orientation of the progeny cells is discussed.