Phylogeographic analysis of the threatened and endangered superconglutinate-producing mussels of the genus Lampsilis (Bivalvia: Unionidae).

Several species of freshwater unionid mussels in the genus Lampsilis exhibit a remarkable reproductive strategy. Female mussels of these species enclose their larvae in a minnow-like lure, called a 'superconglutinate', to attract piscivorous fishes. When a fish attempts to ingest the superconglutinate the lure ruptures and the larvae are released to parasitize the fish. Of the four species of mussel which exhibit this strategy and are endemic to the Gulf Coast drainages of the southeastern United States, three are protected under the Endangered Species Act, and one is recognized as imperiled. Phylogenetic analysis of nucleotide sequences of the mitochondrial 16S ribosomal RNA and the first subunit of the cytochrome oxidase c genes was conducted on 18 individual specimens representing these four species and six outgroup taxa. Phylogenetic analyses of these data support the monophyly of the superconglutinate-producing mussels, and indicates a strong geographical component to the data. The zoogeographic patterns of the four taxa included in the study are congruent with those seen in freshwater vertebrates, and are consistent with a vicariant pattern resulting from fluctuations in sea level during the Pleistocene. Despite the strong geographical structuring of the data, only one species, Lampsilis subangulata, was recovered as monophyletic. The authors attribute the lack of support for the monophyly of the remaining species to insufficient sequence variation and the recent origin of the ancestor of these taxa. Based on these data, any future captive breeding projects aimed at augmenting or re-establishing populations should do so only from the appropriate source populations so as to maintain the genetic integrity of these nascent species.

Neurotrophic factors prevent ceramide-induced apoptosis downstream of c-Jun N-terminal kinase activation in PC12 cells.

Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.

Arachidonic acid activates NADPH oxidase by a direct, calmodulin-regulated mechanism.

In intact neutrophils, arachidonic acid rapidly and transiently activated NADPH oxidase-mediated superoxide generation. Inhibitors of protein kinases (staurosporine and H-7) failed to markedly attenuate arachidonic acid-stimulated superoxide generation. Conversely, the calmodulin antagonist W-7 blocked this arachidonic acid-stimulated response. Similarly, diphenylene iodonium potently inhibited superoxide release. These results suggest that arachidonic acid directly activates the membrane-associated NADPH oxidase and calmodulin and/or calmodulin-binding proteins are required for the assembly of the active oxidase.

A molecular phylogeny of Mobile River drainage basin pleurocerid snails (Caenogastropoda: Cerithioidea).

Sequences from the mitochondrial 16S rRNA gene were obtained to construct a molecular phylogeny for Mobile River drainage basin pleurocerid snails. Data from 876 aligned positions generated a single most-parsimonious tree for each of three analytical approaches: (1) equal weighting, (2) transversions weighted 2 x transitions; and (3) transversions weighted 4 x transitions. Identical topologies for the resulting trees depict the genera Elimia and Pleurocera as monophyletic sister taxa. The genus Leptoxis is paraphyletic with Leptoxis plicata sister to the Elimia + Pleurocera clade. L. taeniata and L. ampla are sister taxa and L. picta is the most basal pleurocerid examined. When transversions were weighted 10x transitions a single most-parsimonious tree was obtained with the only topological difference being L. picta depicted as sister to L. taeniata and L. ampla and L. plicata is now the most basal pleurocerid examined. Many of the Elimia species are closely related, but we await further data before making any taxonomic recommendations. L. picta and L. plicata are quite distinct from each other and all other pleurocerid species examined. These data serves as an important foundation for future studies examining conservation genetics and systematics of this diverse and imperiled family.

Ceramide induces apoptosis in PC12 cells.

The novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C2-ceramide in a dose- and time-dependent manner. Natural ceramide and C6-ceramide had a similar effect. This response was specific since the structural analog C2-dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin-1beta converting enzyme (ICE)-like proteases, Z-VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE-like protease activation in ceramide-induced apoptosis in PC12 cells.

Glycogen metabolism in the rat visceral yolk sac. 2. Activity of glycogen-degrading enzymes.

In an attempt to explain the previous observation of the rise and subsequent fall in glycogen content of the rat visceral yolk sac during the latter half of gestation, the activities of glycogen phosphorylase, glucose-6-phosphatase and lysosomal alpha-glucosidase were measured. Glycogen phosphorylase was found to be present in the yolk sac and, as in adult rat liver, was predominantly in the 'a' (active) form. The specific activity of the enzyme was lower than in adult rat liver, when expressed per mg tissue protein or per mg tissue wet weight, but similar when expressed per mg tissue glycogen. Phosphorylase activity in yolk sac was similar at 16.5 and 18.5 days of gestation. Glucose-6-phosphatase activity was not detectable in the yolk sac at either 15.5 or 18.5 days of gestation. Two lysosomal enzymes, acid alpha-glucosidase and N-acetyl-beta-hexosaminidase, were shown to be present in the yolk sac at higher specific activity than in adult liver. Alpha-Glucosidase activity in yolk sac was similar at 15.5 and 18.5 days of gestation. It is concluded that the net degradation of yolk sac glycogen initiated around 18.5 days of gestation does not serve to provide glucose for the fetus, and may indicate an increased demand for metabolic energy within the yolk sac itself.

Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

Beta 1 integrin-mediated T cell adhesion is regulated by calcium ionophores and endoplasmic reticulum Ca(2+)-ATPase inhibitors.

Treatment of T lymphoblasts with stimuli that mobilize [Ca2+]i, such as ionophores (ionomycin and A23187) and endoplasmic reticulum Ca(2+)-ATPase inhibitors (thapsigargin, 2,5-di-(tert.-butyl)-hydroquinone and cyclopiazonic acid), activated T cell binding to extracellular matrix (ECM) proteins. T lymphoblast adhesion to ECM proteins stimulated by ionomycin, thapsigargin, or PMA was inhibited by an anti-beta 1 integrin mAb (4B4), confirming the role of beta 1 integrins in regulated T cell-ECM interactions. Study of the alpha integrin subunit specificity of activated lymphoblast-fibronectin interactions demonstrated that alpha 5 beta 1 was the major integrin receptor regulating binding to fibronectin. These results indicate that intracellular Ca2+ mobilization plays a major contributory role in the activation of T cell beta 1 integrins.

Fluoride-mediated activation of the respiratory burst in electropermeabilized neutrophils.

Electropermeabilization creates small pores in the plasma membrane allowing the introduction of low-molecular-weight modulatory components, such as ions and nucleotides, into the cytosol. The present study investigates fluoride-mediated stimulation of the signal transduction pathway that activates the respiratory burst in electropermeabilized neutrophils. In marked contrast to intact (i.e., non-electropermeabilized) neutrophils, cells permeabilized by this technique demonstrated an immediate and potent stimulation of the superoxide (O2-)-generating NADPH oxidase in response to the addition of fluoride. Furthermore, permeabilization of neutrophils in the presence of exogenously added ATP enhanced the rate of F(-)-mediated O2- production. Fluoride-stimulated O2- production in electropermeabilized neutrophils was antagonized by GDP beta S and dependent upon the presence of Mg2+ in the medium, but was insensitive to pertussis toxin treatment, consistent with the hypothesis that fluoride activates a G protein, probably Gp, by interacting with the nucleotide-binding site on the G alpha subunit. In addition, electropermeabilized neutrophil O2- release triggered by F- was blocked by staurosporine and H-7, indicating that this pathway proceeds largely through protein kinase C activation. However, nucleotide-enhanced O2- production was only partially blocked by these inhibitors, suggesting that under such conditions ATP either competes with the inhibitor-protein kinase interaction or affects the signaling pathway(s) in such a way that protein kinase C may no longer be necessary for the activation of NADPH oxidase.

Mg.ATP primes superoxide-generating responses in electropermeabilized neutrophils.

The present study utilizes an electropermeabilized cell system to determine the effect of Mg.ATP on neutrophil superoxide (O2-)-generating responses stimulated by suboptimal concentrations of fMLP, GTP gamma S and PMA. Permeabilization in the presence of exogenously added Mg.ATP was neither sufficient to initiate O2- release nor necessary for stimulated O2- production. However, the inclusion of Mg.ATP in the permeabilization medium primed the O2(-)-generating responses mediated by suboptimal concentrations of these stimuli. The site of action of Mg.ATP is intracellular. Moreover, the fact that Mg.ATP primes responses stimulated by fMLP, GTP gamma S and PMA suggests that the modulatory effect is at the level of protein kinase C.

Glycogen metabolism in the rat visceral yolk sac. I. Glycogen content and gestational age.

The glycogen content of the rat visceral yolk sac was determined between 13.5 and 20.5 days of gestation by the best available colorimetric method. The concentration of glycogen in the tissue increased ten-fold between 13.5 and 18.5 days, to reach a value similar to that for mammalian muscle, but then decreased by 50 per cent between 18.5 and 20.5 days. Determination of the iodine-iodide spectra and fractionation of the glycogen particles by a novel sodium citrate centrifugation method indicated broad similarities between the structures of glycogen particles, isolated by a mild phenol-water method, from the yolk sac and the liver of the rat. However, the proportion of 'high'-molecular-weight glycogen in the yolk sac increases between 18.5 and 20.5 days, as a result of the preferential loss of 'low'-molecular-weight glycogen, so that at term the proportion approaches that found in liver glycogen.