Secretory trafficking in neuronal dendrites

The neuronal secretory pathway represents the intracellular route for proteins involved in synaptic transmission and plasticity, as well as lipids required for outgrowth and remodelling of dendrites and axons. Although neurons use the same secretory compartments as other eukaryotic cells, the enormous distances involved, as well as the unique morphology of the neuron and its signalling requirements, challenge canonical models of secretory pathway organization. Here, we review evidence for a distributed secretory pathway in neurons, suggest mechanisms that may regulate secretory compartment distribution, and discuss the implications of a distributed secretory pathway for neuronal morphogenesis and neural-circuit plasticity.     

Triamterene-β-cyclodextrin systems: Preparation,characterization and in vivo evaluation

The purpose of this research was to improve the solubility and therefore dissolution and bioavailability of triamterene, a poorly water soluble diuretic, by complexation with β-cyclodextrin. Triamterene has been reported to show low bioavailability after oral administration, with wide intersubject variation. This study presents the formulation of solid dispersions of triamterene with β-cyclodextrin—by cogrinding, kneading, and coevaporation, using low pH conditions—and their characterizations, evaluation of improvement in dissolution profiles, and in vivo advantage. Phase solubility studies indicated complex with possible stoichiometry of 1∶1 and a stability constant of 167.67M⁻¹. The solid dispersions were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, x-ray diffraction, and differential scanning calorimetry studies. The characterization studies confirmed inclusion of the phenyl ring of triamterene within the nonpolar cavity of β-cyclodextrin in the coevaporate. Remarkable improvement in in vitro drug release profiles in 0.1 N HCl and pH 6.8 phosphate buffer was observed with all dispersions, especially the coevaporate. The coevaporate, when administered orally in rats, also exhibited improved in vivo activity, as measured by net sodium ion excretion, as compared with triamterene powder. Thus, coevaporation of the drug and β-cyclodextrin from acidified alcohol provide the optimum condition for inclusion complexation to give a binary system with remarkable improvement in in vitro drug release profile and in vivo performance.     

Cardiac effects of burn injury complicated by aspiration pneumonia-induced sepsis

Early fluid resuscitation, antimicrobials, early excision, and grafting have improved survival in the early postburn period; however, a significant incidence of pneumonia-related sepsis occurs after burn injury, often progressing to multiple organ failure. Recent studies have suggested that this initial injury (burn injury) primes the subject, producing an exaggerated response to a second insult, such as pneumonia-related sepsis. We developed an experimental animal model that included a third-degree burn over 40% of the total body surface area, followed by sepsis (intratracheal administration of Streptococcus pneumoniae, 4 x 106 colony-forming unit), which was produced either 48 or 72 h after burn injury in adult male rats. Hearts harvested after either burn alone, sepsis alone, or burn plus sepsis were used to assess either contractile function (Langendorff) or cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-10 (ELISA). Experimental groups included the following: 1). sham (sham burn and no sepsis); 2). burn injury alone studied either 24, 48, or 72 h postburn; 3). pneumonia-related sepsis in the absence of burn injury; and 4). pneumonia-induced sepsis studied either 48 or 72 h after an initial burn injury. Burn injury alone (24 h) or sepsis alone produced myocardial contractile defects and increases in pro- and anti-inflammatory cytokine secretion by cardiomyocytes. Sepsis that occurred 48 h postburn exacerbated the cardiac contractile defects seen with either burn alone or sepsis alone. Sepsis that occurred 72 h postburn produced contractile defects resembling those seen in either burn alone or sepsis alone. In conclusion, our data suggest that burn injury primes the subject such that a second insult early in the postburn period produces significantly greater cardiac abnormalities than those seen with either burn alone or sepsis alone.     

Phylogenetic Analysis of New Plant Myosin Sequences

We have sampled a large number of plant taxa, ranging from brown algae to angiosperms, for the presence of myosin sequences. Using phylogenetic analysis, we show that all but two of the new plant myosin sequences fall into two of three preexisting myosin classes. We identified two outlying sequences, which do not fall into any preexisting myosin class. Additionally, all genomic sequences encoding class XI myosins contain an intron in the region studied, suggesting that this genomic region has been conserved over at least 1 billion years of plant evolution. With these data, we can rapidly and consistently classify partial myosin sequences from plants. Our data show that plant myosins do not have clear orthologues in other kingdoms, providing interesting insights into the diversification of myosins.     

Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes

Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.     

Effects of season-dependent irradiance levels and nitrogen-deficiency on photosynthesis and photoinhibition in field-grown rice (Oryza sativa)

Photoinhibition and acclimation of photosynthesis in rice plants grown under N-sufficient (NS) and N-deficient (ND) field conditions were investigated during the tropical wet (WS) and dry (DS) seasons in the Philippines. Diurnal patterns of CO₂ assimilation were examined. There was a transient peak in CO₂ assimilation in the leaves of the NS plants in the early morning during the DS and the WS, which was not seen in the ND plants in either season. ND leaves had lower Ribulose bisphosphate carboxylase/oxygenase (Rubisco) contents and lower chlorophyll contents. A lowered quantum yield of photosystem II (φPSII) was observed in the ND plants at an intermediate irradiance though no differences between N treatments were seen at high irradiance. Analysis of carotenoids indicated a small increase in the de-epoxidation state of the xanthophyll cycle (DES) at mid-day in the ND leaves compared to NS. Photoinhibition was greater in ND leaves when incident mid-day irradiance was increased by altering the leaf angle. Although Rubisco contents were lower in ND plants, photosynthesis in situ did not decline proportionally. For NS plants, Chlorophyll content, but not Rubisco content, was season-dependent and results are discussed in terms of the interaction between irradiance use and N content of rice leaves.     

Identification of the molecular mechanisms contributing to polarized trafficking in osteoblasts

The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion.     

Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation

The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching.