A-signalling and the cell density requirement for Myxococcus xanthus development.

Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.     

Genes required for developmental signalling in Myxococcus xanthus: three asg loci.

asg-carrying strains of Myxococcus xanthus arose in a selection for mutants defective in cell-cell signalling during fruiting body development. All 15 asg mutations examined were found to lie in one of three genetic loci, asgA, asgB, or asgC. The loci were defined by linkage to different insertions of transposon Tn5 and molecular cloning of asgA. asg mutants of all three types were deficient in the aggregation of cells into mounds of the sort that normally give rise to fruiting bodies. asg mutants were also deficient in spore formation; sporulation is normally one of the last steps in fruiting body development. Consistent with a requirement for cell-to-cell signalling, at 1 to 2 h asg+-carrying cells release a material called A-factor that can rescue development of asg mutants. asgA, asgB, and asgC mutants released 5% or less of the asg+ level of A-factor, as measured by bioassay. The experimental results are consistent with the hypothesis that a deficiency in A-factor production or release is the primary developmental defect in asg mutants and that aggregation and sporulation depend on A-factor. asg mutations at all three loci also changed the color and morphology of growing colonies, and failure to release A-factor may itself arise from a defect in growing cells.     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that includes fruiting body formation. One of the first stages in development has been called A-signalling. The asg (A-signalling) mutants have been proposed to be deficient in producing A-signal, resulting in development arresting at an early stage. In this paper, we report the identification of a new asg locus asgD. This locus appears to be involved in both environmental sensing and intercellular signalling. Expression of asgD was undetected during vegetative growth, but increased dramatically within 1 h of starvation. The AsgD protein is predicted to contain 773 amino acids and to be part of a two-component regulatory system because it has a receiver domain located at the N-terminus and a histidine protein kinase at the C-terminus. An asgD null mutant was defective in fruiting body formation and sporulation on CF medium. However, the defects of the mutant were complemented extracellularly when cells were mixed with wild-type strains or with bsgA, csgA, dsgA or esgA mutants, but were not complemented extracellularly by asgA, asgB or asgC mutants. In addition, the mutant was rescued by a subset of A-factor amino acids. Surprisingly, when the mutant was plated on stringent starvation medium rather than CF, cells were able to form fruiting bodies. Thus, it appears that AsgD is directly or indirectly involved in sensing nutritionally limiting conditions. The discovery of the asgD locus provides an important sensory transduction component of early development in M. xanthus.     

Electrophoretic mobility of Alzheimer's amyloid-beta peptides in urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.     

Stimulatory effects of peptides on growth of the free-living nematode Caenorhabditis briggsae.

Evidence is presented that peptide products of hydrolysis of casein, including some di- and tripeptides, but not the constituent amino acids, can stimulate growth of C. briggsae in defined basal medium supplemented with cytochrome C and B-sitosterol. Peptide activity may raise from amino acid imbalances in the medium which causes competitive inhibition of uptake of essential amino acids. Such activity precludes facilitation of heme transport as the sole function of growth factor in C. briggsae. This is the first report in a nutritional role of peptides in invertebrate metazoa.     

Remarkably simple sequence requirement of the M-factor pheromone of Schizosaccharomyces pombe

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMC(Far)-OCH(3)) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.     

Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides

The innate immunity factor lactoferrin harbours two antimicrobial moieties, lactoferricin and lactoferrampin, situated in close proximity in the N1 domain of the molecule. Most likely they cooperate in many of the beneficial activities of lactoferrin. To investigate whether chimerization of both peptides forms a functional unit we designed a chimerical structure containing lactoferricin amino acids 17-30 and lactoferrampin amino acids 265-284. The bactericidal activity of this LFchimera was found to be drastically stronger than that of the constituent peptides, as was demonstrated by the need for lower dose, shorter incubation time and less ionic strength dependency. Likewise, strongly enhanced interaction with negatively charged model membranes was found for the LFchimera relative to the constituent peptides. Thus, chimerization of the two antimicrobial peptides resembling their structural orientation in the native molecule strikingly improves their biological activity.     

Utilization of dipeptides by Lactococcus lactis ssp. cremoris

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000     

The influence of conditioned media and nonessential amino acid supplementation on the growth of cells in vitro

The growth enhancing effect of media conditioned by cells from established lines (BHK and L60) is comparable to that of media conditioned by cells of primary origin (chick embryo). However, the properties of the conditioned media from these two systems show marked differences: the growth enhancing factors in the former are dialyzable and heat-stable, in contrast to the non-dialyzable and heat-labile factors in the latter. Media conditioned for only four hours by BHK or L60 cells stimulated cell growth. Amino acid analyses revealed that non-essential amino acids had appeared in these conditioned media. To verify this as the metabolic basis of conditioning by cells from established lines, media containing dialyzed serum were supplemented with each of six non-essential amino acids, and assayed on BHK and L60 at various population densities. Serine was the most stimulatory and alanine the most inhibitory of the amino acids tested. Mixed supplementation of the medium showed that when low levels of alanine and serine were added simultaneously, cell growth was enhanced but any increase in the level of alanine required an increase in the level of serine also to achieve growth stimulation.     

Fungicidal Activity of Oxadiazolyl Sulfides

Glycyl-l-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases, Bestatin competitively inhibited dipeptide hydrolase activities in isolated cells with a K_i value of 10^?8 m, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10^?4 m bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.     

Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993). We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [3H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides.     

Regulation of the 26S proteasome activities by peptides mimicking cleavage products

The implication of the released peptides in allosteric effects during protein degradation catalyzed by the proteasome is an important question not completely resolved. We present here data showing modulation of 26S proteasome activities by peptides composed of 5 or 6 natural amino acids that mimic the products generated during protein breakdown. Several of these peptides inhibit the chymotrypsin-like activity of the Xenope 26S proteasome whereas its trypsin-like activity is enhanced. The basic peptides produced competitive inhibition of the chymotrypsin-like activity and the acidic peptides, parabolic inhibition involving two different binding sites. Our results are in agreement with a model involving hypothetical non-catalytic sites interacting with effectors to modulate the peptidase activities of the proteasome. They also suggest that allosteric effects may occur in the proteasome during protein degradation.     

C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin.

Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

The structure of l-CB3, a cyanogen bromide fragment from the central portion of the l chain of rat collagen. The tryptic peptides from skin and dentin collagens

The mixture of peptides released by tryptic hydrolysis of the collagen CNBr peptide, αl-CB3, has been resolved by ion-exchange chromatography. The resultant eleven tryptic peptides ranged in size from 3 to 46 amino acids and accounted for all the amino acids of the parent CNBr peptide. Two of the lysines in αl-CB3 from rat dentin collagen were shown to be hydroxylated to a substantial degree by isolation of the appropriate hydroxylysine-containing tryptic peptides. An analysis of the tryptic peptides indicated that αl-CB3 from dentin collagen is identical in structure to that from skin collagen, if hydroxylysine and hydroxyproline are considered equivalent to lysine and proline, respectively.     

A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus

The Myxococcus xanthus asg genes ( asgA, asgB , and asgC ) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG , and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD , the gene encoding the major sigma factor of M. xanthus . The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A-signal restores developmental expression of an A-signal-dependent gene (Ω4521) in the asgC767 ( rpoDEK598 ) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.     

Relative natriuretic activities of synthetic atriopeptins I, II and III

Recent evidence indicates that mammalian atria contain a series of peptides which possess potent natriuretic activity. Using a sensitive and reproducible bioassay developed by this laboratory, the natriuretic and diuretic activities of three peptides, atriopeptins I, II, and III (21, 23 and 24 amino acids, respectively) were compared. Bioassays were conducted in pentobarbital-anesthetized male Sprague-Dawley rats weighing 300-350 grams. At doses ranging from 0.33 to 3.0 micrograms, no significant differences in natriuretic or diuretic activities were observed between the three peptides. The time courses of the natriuretic and diuretic responses to these peptides were also identical. The finding that atriopeptin I (21 amino acids) possesses natriuretic activity equal to that of atriopeptins II and III suggests that the C-terminus residues of atriopeptin III (Phe-Arg-Tyr) are not necessary for full expression of natriuretic activity. However, since several previous reports have indicated that atriopeptin I is considerably less potent as a natriuretic than we report here, perhaps a cautionary note should be sounded concerning the conditions required to produce or to retain full biologic activities of the synthetic atriopeptins.