Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x

Ras proteins are signal transducers for many cellular responses. However, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-RasR12-transformed Rat1 fibroblasts using tetracycline operator/repressor (TetO/TetR)-based conditional vectors. Rat1/TetO-RasR12 (Rat1-Ras) cells produced high levels of H-RasR12 protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ras cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis correlated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not commit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results establish a direct role for Ras in apoptosis, and suggest a functional relationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apoptosis.     

Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.     

Role of residues 230 and 236 of actin in myosin-ATPase activation by actin-tropomyosin

The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.     

Partitioning of lipidated peptide sequences into liquid-ordered lipid domains in model and biological membranes.

We have used a fluorescence assay and detergent fractionation to examine the partitioning of different fluorescent lipidated peptides, with sequences and lipid substituents matching those found in various classes of lipidated cellular proteins, into liquid-ordered (raft-like) domains in lipid bilayers. Peptides incorporating isoprenyl groups, or multiple unsaturated acyl chains, show negligible affinity for liquid-ordered domains in mixed-phase liquid-ordered/liquid-disordered (l(o)/l(d)) bilayers composed of dipalmitoylphosphatidylcholine, a spin-labeled unsaturated phosphatidylcholine, and cholesterol. By contrast, peptides incorporating multiple S- and/or N-acyl chains, or a cholesterol residue plus an N-terminal palmitoyl chain, show significant partitioning into liquid-ordered domains under the same conditions. Interestingly, the affinity of a lipidated peptide for l(o) domains can be strongly influenced, not only by the structures of the lipid substituents but also by the nature and the positions of their attachment to the peptide chain. These results are well correlated with those obtained from parallel assays based on low-temperature detergent fractionation. Using the latter approach, we further demonstrate that a truly minimal l(o) domain partitioning motif [myristoylGlyCys(palmitoyl)-] can mediate efficient incorporation into the "raft" fraction of COS-7 cell membranes.     

Tidal and seasonal effects on survival rates of the endangered California clapper rail: does invasive Spartina facilitate greater survival in a dynamic environment?

Invasive species frequently degrade habitats, disturb ecosystem processes, and can increase the likelihood of extinction of imperiled populations. However, novel or enhanced functions provided by invading species may reduce the impact of processes that limit populations. It is important to recognize how invasive species benefit endangered species to determine overall effects on sensitive ecosystems. For example, since the 1990s, hybrid Spartina (Spartina foliosa × alterniflora) has expanded throughout South San Francisco Bay, USA, supplanting native vegetation and invading mudflats. The endangered California clapper rail (Rallus longirostris obsoletus) uses the tall, dense hybrid Spartina for cover and nesting, but the effects of hybrid Spartina on clapper rail survival was unknown. We estimated survival rates of 108 radio-marked California clapper rails in South San Francisco Bay from January 2007 to March 2010, a period of extensive hybrid Spartina eradication, with Kaplan–Meier product limit estimators. Clapper rail survival patterns were consistent with hybrid Spartina providing increased refuge cover from predators during tidal extremes which flood native vegetation, particularly during the winter when the vegetation senesces. Model averaged annual survival rates within hybrid Spartina dominated marshes before eradication (? = 0.466) were greater than the same marshes posttreatment (? = 0.275) and a marsh dominated by native vegetation (? = 0.272). However, models with and without marsh treatment as explanatory factor for survival rates had nearly equivalent support in the observed data, lending ambiguity as to whether hybrid Spartina facilitated greater survival rates than native marshland. Conservation actions to aid in recovery of this endangered species should recognize the importance of available of high tide refugia, particularly in light of invasive species eradication programs and projections of future sea-level rise.     

Transforming growth factor-beta(1) regulates differentiation of porcine granulosa cells in vitro

Transforming growth factor-beta (TGF-beta) is a potential regulator of ovarian function and follicular development. It is speculated that TGF-beta mediates the events in the follicle which culminate in ovulation of the oocyte. The complex processes which ultimately leads to this natural phenomenon must involve interactions between the 2 major follicular cell types, theca and granulosa cells, and the oocyte. Furthermore, a complex local regulatory system must exist to determine which follicles should undergo development and, eventually, which of those should ovulate or undergo atresia. To begin to understand this perplexing process, we must first understand the variables which control the function of each individual cell type. This study investigated the effect of TGF-beta(1) on FSH-induced porcine granulosa cell differentiation in vitro. Transforming growth factor-beta(1) was shown to inhibit progesterone production at high concentrations (0.1 and 10.0 ng/ml) after 12-, 24- and 48-hour treatment. However, TGF-beta(1) produced a biphasic effect on FSH-induced progesterone production during the 12-hour interval between the 36- and 48- hour treatment periods; TGF-beta(1) stimulated progesterone production at a low concentration (0.001 ng/ml) and inhibited production at high concentrations (0.1 and 10.0 ng/ml). The results obtained from the biphasic effect were not observed during any of the other incubation periods or intervals investigated. These results show that TGF-beta(1) has opposing effects on the differentiation of porcine granulosa cells as compared with those on rat granulosa cells. Moreover, TGF-beta(1) can produce opposing effects within the porcine granulosa cell itself which are specific to the concentration and treatment period used. The results of this study seem to suggest that TGF-beta(1) is species- and time-specific in its regulatory actions on FSH-induced porcine granulosa cell differentiation.     

Direct atomization atomic absorption spectrometry for intact biological samples: application to trace metal analysis in silkworm eggs and their hatched worms.

Highly sensitive carbon rod atomic absorption for trace elements was observed by direct atomization of samples with no pretreatment for intact silkworm eggs, their hatched worms, or National Bureau of Standards biological samples (orchard leaves and bovine liver). Crucial temperatures for drying, ashing, and atomization processes were found to obtain accurate results. Results were consistent with those of activation analyses and of ordinary atomic absorption with wet ashing. The precision and dynamic range of this method were evaluated. The advantages and disadvantages of the present approach were critically discussed.