Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.     

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.     

Revision of <Emphasis Type="Italic">Neolebouria</Emphasis> Gibson, 1976 (Digenea: Opecoelidae), with <Emphasis Type="Italic">Trilobovarium</Emphasis> n. g., for species infecting tropical and subtropical shallow-water fishes

A new opecoelid trematode is reported from fishes of the Lethrinidae, Lutjanidae and Nemipteridae off Lizard Island on the northern Great Barrier Reef, Australia. The new species keys to Neolebouria Gibson, 1976 and shows strong similarity to several species of that genus, but is not consistent with the type-species, N. georgiensis Gibson, 1976, or others known from temperate/polar and/or deep-sea fishes. The new species is also phylogenetically distant from N. lanceolata (Price, 1934) Reimer, 1987, the only representative of the genus for which molecular data are available. A new genus, Trilobovarium n. g., is proposed for the new species, T. parvvatis n. sp. Eight morphologically similar species, previously recognised as belonging to Neolebouria, from shallow-water, mostly tropical/subtropical fishes, are transferred to Trilobovarium: T. diacopae (Nagaty & Abdel Aal, 1962) n. comb.; T. ira (Yamaguti, 1940) n. comb.; T. khalili (Ramadan, 1983) n. comb.; T. krusadaiense (Gupta, 1956) n. comb.; T. lineatum (Aken’Ova & Cribb, 2001) n. comb.; T. moretonense (Aken’Ova & Cribb, 2001) n. comb.; T. palauense (Machida, 2014) n. comb.; and T. truncatum (Linton, 1940) n. comb. Paramanteriella Li, Qiu & Zhang, 1988 is resurrected for five species of Neolebouria with a post-bifurcal genital pore: P. cantherini Li, Qiu & Zhang, 1988; P. capoori (Jaiswal, Upadhyay, Malhotra, Dronen & Malhotra, 2014) n. comb.; P. confusa (Overstreet, 1969) n. comb.; P. leiperi (Gupta, 1956) n. comb.; and P. pallenisca (Shipley & Hornell, 1905) n. comb. Neolebouria georgenascimentoi Bray, 2002, a species with an exceptionally long cirrus-sac, is transferred to Bentholebouria Andres, Pulis & Overstreet, 2004 as B. georgenascimentoi (Bray, 2002) n. comb., and N. maorum (Allison, 1966) Gibson 1976, an unusual species known from cephalopods, is designated a species incertae sedis. Eleven species are retained in a revised concept of Neolebouria.     

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 10⁴ bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.     

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at −80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.     

Elevated CO2 and O3 effects on fine-root survivorship in ponderosa pine mesocosms

Atmospheric carbon dioxide (CO₂) and ozone (O₃) concentrations are rising, which may have opposing effects on tree C balance and allocation to fine roots. More information is needed on interactive CO₂ and O₃ effects on roots, particularly fine-root life span, a critical demographic parameter and determinant of soil C and N pools and cycling rates. We conducted a study in which ponderosa pine (Pinus ponderosa) seedlings were exposed to two levels of CO₂ and O₃ in sun-lit controlled-environment mesocosms for 3 years. Minirhizotrons were used to monitor individual fine roots in three soil horizons every 28 days. Proportional hazards regression was used to analyze effects of CO₂, O₃, diameter, depth, and season of root initiation on fine-root survivorship. More fine roots were produced in the elevated CO₂ treatment than in ambient CO₂. Elevated CO₂, increasing root diameter, and increasing root depth all significantly increased fine-root survivorship and median life span. Life span was slightly, but not significantly, lower in elevated O₃, and increased O₃ did not reduce the effect of elevated CO₂. Median life spans varied from 140 to 448 days depending on the season of root initiation. These results indicate the potential for elevated CO₂ to increase the number of fine roots and their residence time in the soil, which is also affected by root diameter, root depth, and phenology.     

Liberal and Conservative Protestant Denominations as Different Socioecological Strategies

It is common to portray conservative and liberal Protestant denominations as “strong” and “weak” on the basis of indices such as church attendance. Alternatively, they can be regarded as qualitatively different cultural systems that coexist in a multiple-niche environment. We integrate these two perspectives with a study of American teenagers based on both one-time survey information and the experience sampling method (ESM), which records individual experience on a moment-by-moment basis. Conservative Protestant youth were found to be more satisfied, family-oriented, and sociable than liberal Protestant youth, but also more dependent on their social environment, which is reflected in a deterioration of their mood when they are alone. Liberal Protestant youth appear to have internalized values that remain constant whether in the presence or absence of others. We relate these results to the social scientific literature on liberalism and conservatism and to evolutionary theory as a framework for explaining cultural systems as adaptations to multiple-niche environments.