Microzooplankton grazing of phytoplankton in a tropical upwelling region

We measured grazing by herbivorous zooplankton (<200 μm fraction) in coastal and slope regions of the South Brazil Bight. Using the dilution technique, we performed nine experiments during the austral summer, when nutrient-rich South Atlantic Central Water is present on the shelf, and five during winter. These experiments provide the first estimates of microzooplankton grazing in the western South Atlantic Ocean. Model II regression showed a strong relationship between phytoplankton intrinsic growth rates and grazing, with a slope of 0.64 (±0.28; 95% confidence interval) indicating that microzooplankton grazing could account for the majority of phytoplankton mortality. Both phytoplankton growth and microzooplankton grazing were higher during the summer upwelling season, compared to winter. For the two experiments that were conducted in oligotrophic slope water, grazing accounted for >80% of phytoplankton production. A comparison of incubations with and without added inorganic nutrients showed no consistent stimulation of phytoplankton growth (slope of enriched versus unenriched treatments not significantly different from 1). Estimates from microscopic counts of heterotrophic organisms >10 μm indicated that copepod nauplii comprised the largest share of the microzooplankton biomass (mean 62.4 ± 5.8% SE). Grazing estimates were not correlated with microzooplankton biomass, whether or not nauplii were included, suggesting that most of the grazing was done by nano-sized zooplankton. Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at and is accessible for authorized users. Handling editor: S. Wellekens     

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes.

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.     

IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen

Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25(+)CD4(+) regulatory T (T(reg)) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact T(reg) cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/I(IC)). SJL mice were challenged with proteolipid protein peptide 139-151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/I(IC) greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-gamma but diminished TGF-beta production by T(reg) cells from Salmonella-CFA/I(IC)-treated mice. Adoptive transfer of T(reg) cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25(-)CD4(+) T cells from Salmonella-CFA/I(IC) showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-gamma neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/I(IC)-induced T(reg) cells. Thus, the Salmonella-CFA/I(IC) vaccine elicits T(reg) cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these T(reg) cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.     

"Skittish" Abca2 knockout mice display tremor, hyperactivity, and abnormal myelin ultrastructure in the central nervous system

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein most highly expressed in the central and peripheral nervous system tissues and macrophages. Previous studies indicated its role in cholesterol/steroid (estramustine, estradiol, and progesterone) trafficking/sequestration, oxidative stress response, and Alzheimer's disease. Developmental studies have shown its expression during macrophage and oligodendrocyte differentiation, processes requiring membrane growth. To determine the in vivo function(s) of this transporter, we generated a knockout mouse from a gene-targeted disruption of the murine ABCA2 gene. Knockout males and females are viable and fertile. However, a non-Mendelian inheritance pattern was shown among male progeny of heterozygous crosses. Compared to wild-type and heterozygous littermates, knockout mice displayed a tremor without ataxia, hyperactivity, and reduced body weight; the latter two phenotypes were more marked in females than in males. This sexual disparity suggests a role for ABCA2 in hormone-dependent neurological and/or developmental pathways. Myelin sheath thickness in the spinal cords of knockout mice was greatly increased compared to that in wild-type mice, while a significant reduction in myelin membrane periodicity (compaction) was observed in both spinal cords and cerebra of knockout mice. Loss of ABCA2 function in vivo resulted in abnormal myelin compaction in spinal cord and cerebrum, an ultrastructural defect that we propose to be the cause of the phenotypic tremor.     

Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NR_serine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs. Received: 12 February 1998 / Accepted: 28 May 1998     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.