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PubMed2006

Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.

Active Transport, Cell Nucleus↗
PubMed1981

Hormone-cytoplasmic interactions controlling sperm nuclear decondensation and male pronuclear development in starfish oocytes.

Numerous highly condensed sperm nuclei were present in the cytoplasm of starfish oocytes fertilized at the germinal vesicle stage. Decondensation of sperm nuclei and formation of male pronuclei and asters followed treatment of such fertilized oocytes with meiosis-inducing hormone 1-methyladenine (1-MA). Transformation of sperm nuclei was asynchronous, occurred only after germinal vesicle breakdown (GVBD), and was associated with characteristic clearing of the adjacent cytoplasmic organelles. GVBD and sperm nuclear changes were both inhibited when dinitrophenol was added to previously fertilized GV-intact oocytes. Results demonstrate that polyspermy occurs readily in immature oocytes and that 1-MA plays a key role in establishing the block to polyspermy. Furthermore, the factors necessary for sperm nuclear decondensation and pronuclear development do not exist in the cytoplasm of immature oocytes but arise following dispersal of germinal vesicle contents into the cytoplasm. Thus, absence of embryonic development in fertilized GV-intact oocytes is linked to a lack of both oocyte and sperm nuclear differentiation rather than to a lack of sperm entry into the cytoplasm.

Adenine↗
PubMed1993

Chemotactic peptide-induced cytoplasmic pH changes in incubated human monocytes.

Stimulation of phagocytic leukocytes with chemotactic factors results in transient acidification, followed by alkalinization of the cytosol. Human monocytes are known to alter their functional responses to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in a complex fashion as they mature in vitro to macrophages. To examine the evolution of the cytoplasmic pH (pHi) response of monocytes to fMLP as they mature into macrophages, we incubated cells for 0, 24, 48, and 96 h (Medium-199 + 10% fetal bovine serum; 37 degrees C) and examined pHi using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 1 microM) and a Perkin-Elmer 650/10 spectrofluorimeter (lambda em = 530 nm, lambda ex = 500, 450 nm) as previously described. The resting pHi of fresh (0 h) monocytes was 7.07 +/- 0.16 (SD) and was unchanged after incubation for 24, 48, or 96 h (7.09, 7.11, 7.05, respectively). Cells exhibited an fMLP dose-dependent cytoplasmic acidification, with maximal delta pHi occurring 30-60 s after exposure to 10(-7) M fMLP. The response to fMLP did not change with the duration of incubation and, as with neutrophils, cytoplasmic realkalinization was blocked by dimethylamiloride (20 microM). Incubation with 2-deoxyglucose (10 min, 5 mM), sufficient to inhibit by more than 90% the formyl peptide-stimulated superoxide generation by monocytes, slowed fMLP-induced acidification and abrogated the alkalinization. In addition, monocytes isolated from the blood of a patient with X-linked chronic granulomatous disease (CGD) underwent fMLP-induced acidification that was unmasked further by coincubation with dimethylamiloride, in a manner quantitatively similar to that of normal monocytes, despite the inability of the CGD cells to produce superoxide. The chemotactic factor-induced cytoplasmic pH responses of monocytes/macrophages remained constant as the cells matured in vitro and exhibited a dimethylamiloride-independent acidification and dependent alkalinization, as did the response in neutrophils. The cytoplasmic acidification of these cells thus did not correlate with the cells' production of superoxide and with the concomitant hexose monophosphate shunt activation, as has been suggested for other leukocyte types.

Calcium↗
PubMed2006

Myelin protein zero: mutations in the cytoplasmic domain interfere with its cellular trafficking.

The cytoplasmic domain of myelin protein zero (MPZ), the principal protein of peripheral myelin, undergoes phosphorylation on several serine residues and a tyrosine group that is maximal during peak nerve myelination. Mutations that could affect MPZ phosphorylation cause the inherited neuropathy, Charcot-Marie-Tooth disease Type 1B. To investigate a possible role for phosphorylation in regulation of MPZ trafficking within the cell, we expressed wild-type and mutated MPZ-enhanced green fluorescent protein (GFP) fusion proteins in cultured Schwann-like cells. Whereas wild-type protein is present almost entirely at the cell surface, mutation of serine 204 to alanine or at a nearby presumed PKC substrate motif (198RSTK201) causes 40-60% of protein to be retained in the cytoplasm. Mutation of S204 to aspartate, which introduces a permanent negative charge, also impairs MPZ movement to the plasma membrane. In contrast, tyrosine 191 mutation has no effect on MPZ cellular distribution. Simultaneous alteration of S204 and Y191 produces much less perturbation of MPZ trafficking than mutation of S204 alone. Colocalization studies showed that mutated MPZ-EGFP trapped in the cytoplasm associates with all organelles in the secretory pathway. Previous studies have shown that cytoplasmic mutations at serine, but not tyrosine phosphorylation sites, abolish MPZ adhesive properties. Our results suggest that this loss of adhesion may be due, at least in part, to a failure of sufficient MPZ to reach the cell surface and that this impaired trafficking is associated with deficient serine phosphorylation in the cytoplasmic domain.

Amino Acid Sequence↗
PubMed1977

Interaction between cytoplasmic (Ca2+--Mg2+) ATPase activator and the erythrocyte membrane.

Human red blood cells (RBC) contain a cytoplasmic, nonhemoglobin protein which activates the (Ca2+-Mg2+)ATPase of isolated RBC membranes. Results presented in this paper confirm that activation of (Ca2+-Mg2+)ATPase is associated with binding of the cytoplasmic activator to the membrane. Binding of the cytoplasmic activator is reversible and dependent on ionic strength and Ca2+. Cytoplasmic activator is sensitive to trypsin but is not degraded when intact RBC are exposed to trypsin. Cytoplasmic activator does not modify the (Ca2+-Mg2+)-ATPase of membranes from RBC exposed to activator prior to hemolysis. Thus, the activator is located in the cell and appears to act by binding to the inner membrane surface.

Adenosine Triphosphatases↗
PubMed2003

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes.

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.

Animals↗
PubMed2005

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2.

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.

Amino Acid Sequence↗
PubMed2000

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae.

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.

Amino Acids↗
PubMed1993

Cytoplasmic-nuclear translocation of the Hsp70 protein during environmental stress in Trypanosoma cruzi.

The present study provides immunological evidence of the constitutive presence of the Hsp70 protein in the cytoplasm of logarithmically growing T. cruzi parasites cultured at the normal temperature of 28 degrees C and of the translocation of the protein to the nucleus upon a heat shock treatment (2 hours at 37 degrees C). The nuclear translocation of the protein must depend on other factors beside the temperature per se since at 28 degrees C, in stationary phase growing parasites, the Hsp70 protein was present in both the cytoplasm and the nucleus. During recovery at 28 degrees C the protein leaves the nuclei but the nuclear-cytoplasmic translocation of the protein is a much more gradual process than its initial transport to the nucleus. Since the isoform of the nuclear Hsp70 is different from that found in the cytoplasm it is likely that before translocation to the nucleus the cytoplasmic Hsp70 nuclear precursor must undergo a specific modification.

Animals↗
PubMed2002

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells.

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.

Active Transport, Cell Nucleus↗
PubMed1993

Cleavage asynchrony in the Tubifex embryo: involvement of cytoplasmic and nucleus-associated factors.

The smaller AB-cell of the 2-cell embryo of Tubifex divides 40 min later than the larger CD-cell. The present study was undertaken to elucidate those factors that are responsible for this cleavage asynchrony. An examination of chromosome cycles showed that the AB-cell entered M-phase 5 min later and spent about 35 min longer in the prometaphase portion of M-phase than did the CD-cell. Even if cultured in isolation, the length of M-phase of AB isolates continued to be comparable to that of intact AB-cells. When AB and CD nuclei were brought into a common cytoplasm, they entered M-phase simultaneously. The durations of M-phase portions from prometaphase through telophase were not altered even in a common cytoplasm, as long as the mitotic figures of AB and CD nuclei were separated from each other. Only when the mitotic spindles of both nuclei were united at at least one pole was the length of prometaphase of the AB nucleus significantly shortened. Whole-mount immunocytochemistry of microtubules revealed that asters were involved in mitotic spindle assembly in the CD-cell but not in the AB-cell. The fully formed spindle of the AB-cell was anastral and barrel-shaped, while that of the CD-cell possessed asters at its poles. The morphology of the spindle was not altered by changes in the cytoplasmic environments; that is, the spindle of AB nucleus was found to be anastral even in the presence of a CD-cell cytoplasm. These results suggest that unlike the case of other animals so far studied, cleavage asynchrony in the Tubifex embryo is brought about not only by cytoplasmic components but also by nucleus-associated factors.

Animals↗
PubMed1994

Effects of cytoplasmic components upon sperm aster development in Bufo arenarum eggs.

Bufo arenarum oocytes obtained during the winter period, presenting a metabolism similar to that of the differentiated tissues, are not able to form a sperm aster after spermatozoon injection. These oocytes may be considered immature, with respect to the state of their cytoplasm. In the present work, aster formation was induced in winter coelomic oocytes through injection of cytoplasm from summer oocytes, GTP, and EDTA. When winter oocytes received cytoplasm from summer oocytes, they became able to form asters. If the cytoplasm interchange was inverted, i.e., if the cytoplasm of winter oocytes was injected into summer oocytes, no modification of the capacity to form asters was detected. In addition, the injection of GTP into winter oocytes induced the formation of asters in up to 60% of the oocytes, while the chelation of Ca2+ with EDTA had no effect on aster formation. Winter coelomic oocytes behaved as oocytes during prophase even after germinal vesicle breakdown. From the above, it may be suggested that these oocytes cannot activate the microtubule organizing centers.

Animals↗
PubMed1996

Cytoplasmic polyadenylation of activin receptor mRNA and the control of pattern formation in Xenopus development.

The activin receptor, a transmembrane serine-threonine kinase, is a key component necessary for pattern formation in early Xenopus development. This protein interacts with members of the transforming growth factor beta family and stimulates cells of the marginal zone to differentiate along the mesodermal pathway. In large part, this function of the activin receptor has been inferred from observations of phenotypes induced by injected mRNA encoding wild-type or mutant forms of the protein. Naturally occurring activin receptor mRNA is maternally inherited and contains within its 3' untranslated region an embryonic-type cytoplasmic polyadenylation element (CPE), an oligouridylic acid sequence that promotes cytoplasmic polyadenylation and resultant translational activation. Based on the presence of this element, we predicted in a previous report that activin receptor mRNA expression in embryos might be regulated by cytoplasmic polyadenylation (Simon and Richter, Mol. Cell. Biol. 14, 7867-7875, 1994). In this study, we have tested this hypothesis and show that not only do endogenous and injected activin receptor mRNAs undergo cytoplasmic polyadenylation during embryogenesis, but also that this process is necessary for stimulating translation and inducing the morphological defects observed by mRNA overexpression. The activin receptor CPE is bound by a Mr 36 x 10(3) protein in vitro, and competition for this factor between mRNAs in vivo inhibits activin receptor mRNA polyadenylation. This competition may be responsible for the lack of mesoderm formation observed in such injected embryos. These data suggest that cytoplasmic polyadenylation controls differentiation and pattern formation in early Xenopus development.

Activin Receptors↗
PubMed1997

Cytoplasmically anchored plakoglobin induces a WNT-like phenotype in Xenopus.

Plakoglobin is one of two vertebrate proteins closely related to the Drosophila segment polarity gene product armadillo. Overexpression of plakoglobin induces neural axis duplication in Xenopus and the exogenous plakoglobin is localized to nuclei (Karnovsky, A., and Klymkowsky, M. W., Proc. Natl. Acad. Sci. USA 92, 4255, 1995; Rubenstein, A., et al., Dev. Genet., 1997, in press). We have carried out a series of experiments to test whether the nuclear localization of plakoglobin is required for its inductive effects. Prior to the midblastula transition exogenous plakoglobin is cytoplasmic and concentrated in the cortical regions of blastomeres; after the midblastula transition exogenous plakoglobin accumulates in embryonic nuclei. The addition of a "nuclear localization sequence" does not change the timing of plakoglobin's nuclear localization, suggesting that it is anchored in the cytoplasm prior to the midblastula transition. Next, we constructed two "membrane-anchored" forms of plakoglobin. These are exclusively cytoplasmic; yet both were as effective at producing a "Wnt-like" axis duplication as were "free," unfettered forms of plakoglobin. Moreover, expression of anchored plakoglobins had no apparent effect on the cytoplasmic or nuclear levels of beta-catenin. These data indicate that plakoglobin can act cytoplasmically to generate a WNT-like phenotype. Taken together with the ventralizing effects of a mutant from of the XTcf-3 transcription factor, described by Molenaar et al. Cell 86, 391, 1996, we speculate that in the early Xenopus embryo, activation of plakoglobin (or beta-catenin) inhibits the activity of XTcf-3 or a XTcf-3-like factor.

Amino Acid Sequence↗
PubMed2000

The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif.

Integrins are heterodimeric transmembrane proteins that mediate substrate adhesion and migration but also the bidirectional transfer of information across the plasma membrane via their cytoplasmic domains. We addressed the question of whether the very short cytoplasmic tail of the alpha1 integrin subunit of alpha1beta1 integrin is required for alpha1beta1-specific adhesion, spreading, and migration. For this purpose we transfected the alpha1 integrin subunit and two cytoplasmically truncated alpha1 subunits into Chinese hamster ovary (CHO) cells. Elimination of the entire cytoplasmic domain of the alpha1 subunit does not affect adhesion but leads to inhibition of spreading and stress fiber formation. The defect in spreading could not be rescued by lysophosphatidic acid, which has been reported to stimulate actin stress fiber formation via Rho. Additionally, deletion of the entire cytoplasmic domain of the alpha1 subunit abolishes migration toward alpha1beta1-specific substrates. Migration and stress fiber formation are similar in CHO-alpha1 cells and CHO cells carrying an alpha1 subunit still containing the conserved GFFKR motif. So, the GFFKR motif of the alpha1 subunit is essential and sufficient for these processes.

Actins↗
PubMed2002

Fourier analysis of cytoplasmic texture in nuclear fiber cells from transparent and cataractous human and animal lenses.

Comparisons were made of the cytoplasmic textures in electron microscope images of nuclear fiber cells from a variety of human and animal lenses. The goals were to establish the optimal conditions for quantifying the textural features and for relating the extent of roughness with the observed extent of nuclear opacification. Freshly cut Vibratome sections were fixed and processed for thin-section electron microscopy. Normal human donor lenses, human age-related cataracts from surgery, and rat, guinea pig, and canine lenses were analyzed using density linescans, Fourier transforms, and autocorrelation analysis. Normal and control lenses were compared to lenses with varying degrees of scattering including fully opaque nuclear cataract. Images were recorded at 21,000 x, giving structural information in the critical range of 2-300 nm. Human normal and nuclear cataractous lens cytoplasm produce Fourier transforms with relatively high intensity in the range 10-50 nm (equivalent spacing) and relatively low intensity greater than 100 nm. This is consistent with the smooth image appearance, linescans with small fluctuations and autocorrelation functions indicating that the images are nearly homogeneous. Images of the transparent animal lenses were very smooth and produced Fourier transforms that showed less intensity in the range 10-50 nm and less intensity greater than 100 nm compared to the human lenses. Animal lenses with progressively enhanced light scattering showed a strong correlation between increased textural roughness and increased Fourier intensity greater than 100 nm. These analytical image analysis techniques readily documented the wide range of cytoplasmic textural variations in human and animal lenses and cataracts. Consistent comparisons were possible only when well-preserved tissues were examined with high-resolution images. The cytoplasm with the greatest roughness correlated with the greatest light scattering suggests that redistribution and/or loss of cytoplasmic proteins contribute to cataract formation.

Adult↗
PubMed1998

Expression of an antibody fragment at high levels in the bacterial cytoplasm.

Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm.

Amino Acid Sequence↗
PubMed1997

Cytoplasmic redox potential affects energetics and contractile reactivity of vascular smooth muscle.

Variations in the cytoplasmic redox potential (Eh) and NADH/NAD ratio as determined by the ratio of reduced to oxidized intracellular metabolite redox couples may affect mitochondrial energetics and alter the excitability and contractile reactivity of vascular smooth muscle. To test these hypotheses, the cytoplasmic redox state was experimentally manipulated by incubating porcine carotid artery strips in various substrates. The redox potentials of the metabolite couples [lactate]/[pyruvate]i and [glycerol 3-phosphate]/[dihydroxyacetone phosphate]i varied linearly (r=0.945), indicating equilibrium between the two cytoplasmic redox systems and with cytoplasmic NADH/NAD. Incubation in physiological salt solution (PSS) containing 10 mm pyruvate ([lact]/[pyr]=0.6) increased O2 consumption approximately 45% and produced anaplerosis of the tricarboxylic acid (TCA cycle), whereas incubation with 10 mm lactate-PSS ([lact]/[pyr]i=47) was without effect. A hyperpolarizing dose of external KCl (10 mM) produced a decrease in resting tone of muscles incubated in either glucose-PSS (-0.8+/-0.8 g) or pyruvate-PSS (-2.1+/-0.8 g), but increased contraction in lactate-PSS (1.5+/-0.7 g) (n=12-18, P<0.05). The rate and magnitude of contraction with 80 mm KCl (depolarizing) was decreased in lactate-PSS (P=0.001). Slopes of KCl concentration-response curves indicated pyruvate>glucose>lactate (P<0.0001); EC50 in lactate (29. 1+/-1.0 mM) was less than that in either glucose (32.1+/-0.9 mm) or pyruvate (32.2+/-1.0 mM), P<0.03. The results are consistent with an effect of the cytoplasmic redox potential to influence the excitability of the smooth muscle and to affect mitochondrial energetics.

Aerobiosis↗