Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.     

A tunable switch to regulate the synthesis of low and high molecular weight microbial polyesters

The addition of poly(ethylene glycol) (Mn = 200 g/mol) (PEG-200) to the fermentation media of Alcaligenes eutrophus and Alcaligenes latus at various stages of growth resulted in the synthesis of poly(3-hydroxybutyrate) (PHB) with bimodal molecular weight distributions. The presence of 2% w/v-PEG-200 did not have deleterious effects on PHB volumetric yields and cell productivity. In general, the Mn values of the high (H) and low (L) fractions showed little variability as a function of the time at which PEG-200 was added to the cultures. By this approach, the H:L ratios (w/w) of the PHB synthesized by A. eutrophus and A. latus were varied from 9:91 to 76:24 and from 16:84 to 88:12, respectively. It is believed that the H fractions were formed prior to the addition of PEG-200 to the cultures. Also, once PEG-200 was made available to the cells, PEG-200 acted as a switch so that the reduced molecular weight fraction was formed. In addition, a necessary requirement for the above is that the frequency of transesterification reactions during polymer synthesis was small. The efficiency that PEG-200 reduced the molecular weight of the PHBs formed by both bacteria appears similar. Indirect evidence suggests that the PHB L fractions formed by A. latus subsequent to PEG-200 addition consist primarily of chains that have PEG terminal groups. This terminal chain structure was not observed for PHB formed by A. eutrophus.     

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.     

The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1

The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.     

Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP₂) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP₂ in the cells was 910 ± 100 pmol g⁻¹ fresh weight; by 12 d, PIP₂ levels increased to 1200 ± 150 pmol g⁻¹ fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min⁻¹ mg⁻¹ protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP₃) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP₃ was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP₃ signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP₂ biosynthesis in cells with already-high PIP₂ levels suggests that the PIP₂ present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP₂.     

Biosynthesis of novel exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 x 10(6) to 2.12 x 10(6) to 0.85 x 10(6) to 0.77 x 10(6) with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 +/- 3 to 29 +/- 2 mol%, and the molecular weight increased from 2.73 x 10(6) to 4.86 x 10(6). There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 +/- 3:13 +/- 3 to 28 +/- 2:72 +/- 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.