Synthesis of several membrane proteins during developmental aggregation in Myxococcus xanthus

We have examined the pattern of synthesis of several membrane proteins during the aggregation phase of development in Myxococcus xanthus. Development was initiated by plating vegetative cells on polycarbonate filters placed on top of an agar medium that supported fruiting body formation. At various times during aggregation a filter was removed, the cells were pulse-labeled with [35S]methionine, and the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rate of synthesis of numerous individual proteins changed during aggregation; we concentrated on six whose pattern of synthesis was greatly altered during aggregation. The rate of synthesis of five of the six proteins increased considerably during aggregation; that of the remaining protein was curtailed and appeared to be regulated by nutrient conditions. Three of the five major membrane proteins that increased during aggregation had a unique pattern of synthesis that was displayed only under conditions that are are required for development - high cell density, nutrient depletion, and a solid (agar) surface. The remaining two proteins were not unique to development; the appearance of one protein could be induced under conditions of high cell density, whereas the other could be induced by placing the cells on a solid agar surface. All of the five major proteins that appeared during development did so during the preaggregation stage, and the synthesis of four of the five proteins appeared to be curtailed late in aggregation. The synthesis of the remaining protein continued throughout aggregation.     

Morphogenesis in the slime mold Dictyostelium discoideum. 1. The accumulation and erasure of "morphogenetic information".

When developing cultures of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis immediately reinitiated, they recapitulate the morphogenetic scheme, but at an increased rate. Employing this feature of the system, we have identified time periods when “information” accumulates for specific morphogenetic events. In this case, we have defined “morphogenetic information” as the reduction in time for the appearance of a particular morphology during morphogenetic recapitulation. Accumulated information can be erased by disaggregating developing cultures and reinoculating them into liquid growth medium. Erasure occurs as a discrete event and can be blocked by inhibiting protein synthesis.By comparing morphogenesis in log-phase and stationary-phase cultures, at least two parallel timers have been distinguished, one specific for aggregation and one or more specific for morphogenetic events following aggregation. Approaches to the molecular identity of morphogenetic information and morphogenetic timers are discussed.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanisms of platelet activating factor-induced aggregation and secretion in human platelets

The role of TXA2 in PAF-induced aggregation and secretion of human platelets is unclear. We have studied the relationship between aggregation, synthesis of TXA2 and release of 5-HT during the time course of aggregation induced by PAF and collagen. For PAF-induced aggregation there was strong aggregation and secretion with minimal production of TXA2 in contrast to collagen in which a surge in TXA2 synthesis preceded both aggregation and secretion. To determine the role of calcium flux in PAF-induced aggregation we have similarly studied the temporal relationships between aggregation, secretion and TXA2 synthesis for calcium ionophore A23187 induced aggregation but found these to be distinctly different from those determined for PAF. A method for measuring absolute amounts of 5HT released from platelets in small volumes of plasma is described. We conclude that TXA2 is not important in the mechanism of PAF induced aggregation and that an increase in the level of intraplatelet calcium per se is not sufficient to explain the mediation of PAF-induced aggregation.     

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria

Time-dependent aggregation of a plasmid-encoded β-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 μm³ h⁻¹, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.     

Species-specific aggregation factor in sponges. VII. Its effect on cyclic amp and cyclic gmp metabolism in cells of Geodia cydonium.

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

Regulation of the synthesis of two carbohydrate-binding proteins in Dictyostelium discoideum

The relative rate of de novo synthesis of two membrane-associated carbohydrate-binding proteins (CBP) has been examined during Dictyostelium development. The results show that the relative rate of CBP synthesis is minimal during the vegetative stage and increases to represent approximately 3.5 to 5% of newly synthesized protein during the aggregation stage after which the relative rate decreases. Analysis of the relative rates of synthesis of CBP-26 and CBP-24 indicate that at the peak period of synthesis (approximately 5 to 9 h of development) CBP-26 is synthesized at a rate which is approximately eight times greater than CBP-24. In addition, we have examined the relative amount of CBP-26 and CBP-24 mRNA during development as assayed by its ability to direct CBP synthesis in in vitro protein-synthesizing systems. We show that there is no detectable CBP mRNA in vegetative cells and that during the pre-aggregating stages, assayable CBP mRNA appears and accumulates with a maximal level at the period of peak in vivo CBP synthesis. These results suggest that the rate at CBP synthesis in vivo is controlled by the relative amount of functional mRNA.     

Inhibition of desmosome formation in chick cell aggregates.

Desmosomes (macula adherens) have been associated with the function of adhesion. Their possible role in aggregation and sorting of chick and mouse epithelial cells has been investigated. Treatment of aggregates with 2-5 microgram/ml of actinomycin D which inhibited RNA synthesis also inhibited both desmosome formation and aggregation if administered at the beginning of the aggregation process. In contrast, if the drug was administered at six hours, when the cells had recovered from the process of dissociation, then aggregation over the following six hours appeared normal from observation of living samples. Such aggregates incorporated leucine-3H at roughly 85% of the control level. A quantitative comparison was made of desmosome formation in aggregates treated with actinomycin D for hours 6-12 and those cultured in normal medium. Desmosome formation was inhibited by the drug, although aggregation could proceed. Combinations of chick corneal and mouse skin cells sorted out in the presence of actinomycin D to the same extent as controls. Thus desmosome formation, which normally occurs during aggregation of the epithelial cells studied here, is not coupled with the aggregation or cell sorting process in these cells of stratified epithelia. When cells were treated with cycloheximide (100 muM) both desmosome formation and the progressive rounding up of aggregates was inhibited.     

C-signal: a cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies and sporulation are co-ordinated temporally and spatially. csgA mutants fail to synthesize the cell surface-associated C-signal and are unable to aggregate and sporulate. We report that csgA encodes two proteins, a 25 kDa species corresponding to full-length CsgA protein and a 17 kDa species similar in size to C-factor protein, which has been shown previously to have C-signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies. These results show that C-signal induces aggregation as well as sporulation, and that an ordered increase in the level of C-signalling during development is essential for the spatial co-ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C-signalling. In this model, the two events are temporally co-ordinated by the regulated increase in C-signalling levels during development. The contact-dependent C-signal transmission mechanism allows the spatial co-ordination of aggregation and sporulation by coupling cell position and signalling levels.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

Flagellar adhesion and deadhesion in chlamydomonas gametes: Effects of tunicamycin and observations on flagellar tip morphology

The aggregation-dependent loss of flagellar adhesiveness in Chlamydomonas reinhardi has been correlated with changes in flagellar tip morphology during adhesion and deadhesion. As aggregating mt^? and impotent (able to adhere, but not fuse) mt⁺ gametes begin to disaggregate in the presence of the protein synthesis inhibitor cycloheximide, there is a concomitant change in flagellar tip morphology from the activated bulbous form to the nonactivated tapered shape. The requirement of protein-synthetic activity for the maintenance of flagellar adhesiveness during aggregation may be due in part to turnover of proteins involved in formation or stabilization of activated flagellar tips. Incubation of aggregating gametes with tunicamycin indicates that, like protein synthesis inhibitors, this inhibitor of glycosylation also causes adhering gametes to deadhere. The results suggest that protein glycosylation may be essential for maintenance of adhesiveness during aggregation.     

A modified Arg-Asp-Val (RDV) peptide derived during the synthesis of Arg-Glu-Asp-Val (REDV), a tetrapeptide derived from an alternatively spliced site in fibronectin, inhibits the binding of fibrinogen, fibronectin, von Willebrand factor and vitronectin to activated platelets.

Characterization of a side-product obtained during the synthesis of Arg-Glu-Asp-Val (REDV) with inhibitory activity in thrombin-activated platelet aggregation was carried out. The semipreparative column fractionation of REDV peptide was rechromatographed on an analytical HPLC column and revealed two peaks which were re-tested for inhibitory activity. Using amino acid analysis with sequencing and fast atom bombardment mass spectrometry (FABMS), the first peak was determined to be REDV with molecular mass of 517 Da, and the second peak was determined to be a modified RDV with a mass of 608 Da. The modified RDV peptide inhibited thrombin-induced platelet aggregation with an IC50 of 200 microM, and complete inhibition occurred at 600 microM. However, the REDV peptide did not inhibit platelet aggregation up to 1 mM concentration. The modified RDV peptide eluted platelet glycoprotein IIb-IIIa complex that had been bound to GRGDSP-agarose. These studies show that the modified RDV peptide interacts with the platelet glycoprotein IIb-IIIa complex. Based on the collision-induced dissociation (CID) mass spectral data analysis, the modified RDV peptide has been characterized to contain an N-terminus blocking group on the Arg residue. The origin of this blocking group is presumed to have originated from decomposition products of the phenylacetamidomethyl (PAM) resin used in the solid-phase synthesis of the target peptide Arg-Glu-Asp-Val.     

Tyrosine hydroxylase activity in rat brain following "REM" sleep deprivation

I n R ecent years biogenic amines have been implicated in the control mechanism for induction and maintenance of sleep processes (J ouvet , 1969). Investigators have looked for changes in the rate of synthesis of cerebral norepinephrine from [³H]tyrosine after REM sleep deprivation and reported increased rates of synthesis during REM sleep deprivation (M ark , H einer , M andel and G odin , 1969) and REM sleep rebound following 91 h of deprivation (P ujol , M ouret and G lowinski , 1968). Because tyrosine is thought to be the rate-limiting enzyme (U denfriend , 1966) in the synthetic pathways for norepinephrine and since the above-mentioned studies are suggestive of changes in the activity of the enzyme, we decided to measure tyrosine hydroxylase activity following REM sleep deprivation.     

Implications of protein structure instability: from physiological to pathological secondary structure

Proteins are folded during their synthesis; this process may be spontaneous or assisted. Both phenomena are carefully regulated by the "housekeeping" mechanism and molecular chaperones to avoid the appearance of misfolded proteins. Unfolding process generally occurs during physiological degradation of protein, but in some specific cases it results from genetic or environmental changes and does not correspond to metabolic needs. The main outcome of these phenomena is the appearance of nonfunctional pathologically unfolded proteins with a strong tendency to aggregation. Moreover, for some of these unfolded proteins, the agglomeration that follows initial proteins association may give rise to highly structured soluble aggregates. These aggregates have been identified as the main cause of the so-called amyloidosis or amyloid diseases, such as Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases, and type II diabetes mellitus. Although some common mechanisms of amyloid protein aggregation have been identified, the roles of the environmental conditions inducing amyloidosis remain to be clarified. In this review, we will summarize recent studies identifying the origin of amyloid nucleation and will try to predict the therapeutic prospects that may be opened by elucidation of the amyloidosis mechanisms.     

Cholesterol availability modulates myoblast fusion

The requirement of cholesterol for myoblast fusion has been linked to the primary step in the fusion process, calcium-dependent aggregation (recognition). Inhibition of cholesterol synthesis with 25- hydroxycholesterol or compactin in the absence of exogenous lipid dramatically inhibits calcium-mediated aggregation and concomitant fusion within several hours. Restimulating cholesterol synthesis or supplying exogenous cholesterol rapidly restores aggregation activity. Over this time period, however, the sterol:phospholipid ratio is unaltered, suggesting a local rather than a general membrane cholesterol requirement for the expression of aggregation activity. The aggregation response to a change in sterol availability occurs on a shorter time scale than that required to inhibit the synthesis of the protein(s) with aggregation activity; thus, the cholesterol-requiring step is posttranslational. We suggest that the assembly or maintenance of the aggregation activity depends on a continued local supply of cholesterol.     

Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology

The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.