Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

Influence of coenzyme Q10 on tissue distribution of archaeosomes, and pegylated archaeosomes, administered to mice by oral and intravenous routes.

The biodistribution of orally and intravenously administered archaeosomes in mice was compared to that of archaeosomes containing either coenzyme Q10 (archaeosome-CoQ10), polyethylene glycol (archaeosome-PEG), or PEG plus CoQ10 (archaeosome-PEG-CoQ10). The archaeosome formulations were prepared by a reverse-phase evaporation method using the total polar lipids from the archaeobacterium Methanosarcina mazei. In the case of oral gavage, the most striking observation was that a significantly (p < 0.05) higher concentration (42.28+/-4.17%) of administered dose was found in the stomach content 3 h after administration of unmodified archaeosomes, as compared to that of archaeosome-CoQ10 (16.98+/-2.48%) and archaeosome-PEG-CoQ10 (5.8 +/-4.05"/ vesicles. This correlated with an increased uptake, notably of the archaeosome-PEG-CoQ 0 vesicles.,into liver and spleen; however, no more than 7% of the administered dose was found in liver, spleen and blood at any time point studied. In the case of intravenous administration, a significantly higher percentage of injected dose of unmodified archaeosomes was found in the liver (66.4 +/-.92%) and spleen (11.445+/-.68%) at 48 h, compared to archaeosome-CoQ10, archaeosome-PEG, and archaeosome-PEG-CoQ10 vesicles. The combination of PEG and CoQ10 significantly prolonged the circulation of archaeosomes in the blood, but after 48 h the amount of the vesicle marker in blood had declined to only about 0.5% of administered dose. These data indicate that the biodistribution of archaeosome formulations given orally or intravenously can be altered significantly by incorporating PEG or CoQ 10, alone or in combination, and these vesicles have the potential to act as a carrier for therapeutics and vaccines.

In vitro assessment of archaeosome stability for developing oral delivery systems.

The in vitro stability of archaeosomes made from the total polar lipids of Methanosarcina mazei, Methanobacterium espanolae or Thermoplasma acidophilum, was evaluated under conditions encountered in the human gastrointestinal tract. At acidic pH, multilamellar vesicles (MLV) prepared from T. acidophilum lipids were the most stable, releasing approximately 80, 20, 10 and 5% of encapsulated 14C-sucrose at pH 1.5, 2.0, 2.5 and 6.2, respectively, after 90 min at 37 degrees C. Archaeosomes from M. mazei lipids were the least stable. For each type of total polar lipid, unilamellar vesicles (ULV) were less stable than the corresponding MLV vesicles. Pancreatic lipase had relatively minor effect on the stability of archaeosomes made from either of the three types of total polar lipids, causing the release of 12-27% of the encapsulated 5(6)-carboxyfluorescein (CF) from ULV and MLV after 90 min at 37 degrees C. In simulated human bile at pH 6.2, MLV from M. mazei total polar lipids lost 100% of the encapsulated CF after 90 min at 37 degrees C, whereas those from the polar lipids of M. espanolae or T. acidophilum lost approximately 85% of the marker. Pancreatic lipase and simulated human bile had no synergistic effect on the release of carboxyfluorescein from ULV or MLV prepared from any of the total polar lipids. After 90 min in the combined presence of these two stressors at pH 6.2, the leakage of fluorescein conjugated bovine serum albumin from MLV prepared from T. acidophilum lipids was similar to that of CF, and 13% of the initially present vesicles appeared to be intact. These results indicate that archaeosomes show stability properties indicative of potential advantages in developing applications as an oral delivery system.

A simple apparatus for measuring the Eh of anaerobic media.

A 22-mm outside diameter screw cap test tube was modified for measuring the redox potential (Eh) of samples of anaerobic media. The screw cap was fitted with a grey butyl rubber septum with a hole through which a platinum-calomel combination electrode was inserted. The tube was shortened in length and a small side arm that could be sealed with a serum stopper and an aluminium seal was attached. This device enabled the measurement of Eh of anaerobic media without the need for continuous flushing of the vessel headspace to maintain anaerobiosis.

Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site.

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.

Fermentation of xylose and hemicellulose hydrolysates by an ethanol-adapted culture of Bacteroides polypragmatus.

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v) D-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (mu = 0.30 h-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7-9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.

Inhibition of pure cultures of methanogens by benzene ring compounds.

The inhibition of methane production by Methanosaeta concilii GP6, Methanospirillum hungatei GP1, Methanobacterium espanolae GP9, and Methanobacterium bryantii M.o.H. during short-term (6-h) exposure to eight benzene ring compounds was studied. The concentration that caused 50% inhibition of the methane production rate (IC50) was dependent on the species and the toxicant. Pentachlorophenol was the most toxic of the tested compounds, with an IC50 of less than 8 mg/liter for all species except M. hungatei. Abietic acid was the next most toxic compound for all the species, with an IC50 in the range of 21.4 to 203 mg/liter. Sodium benzoate was generally the least toxic, with an IC50 in the range of 1,225 to 32,400 mg/liter. 3-Chlorobenzoate was substantially more toxic (IC50, 450 to 1,460 mg/liter) than benzoate. The inhibition by benzene, phenol, vanillic acid, and toluene was intermediate to that of pentachlorophenol and benzoate. Long-term incubation (days) studies to determine effect on growth indicated that all eight compounds were usually much more toxic than predicted from the short-term data. In these latter studies, there was generally a good correlation in the observed inhibition as determined from growth and methane production.

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts.

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mug of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mug of vector DNA. However, a 380-fold improvement (3,417 transformants per mug of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade.

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.

Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.

The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye.