Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-'lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators ofglgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.A large body of evidence shows that during the transition into stationary phase, bacteria acquire numerous new physiological properties which enhance their ability to compete and survive under suboptimal conditions (for reviews, see references 16, 25, 26, and 44). While it has become clear that in Escherichia coli the induction of several genes and operons in the stationary phase requires a putative sigma factor, katF or rpoS (2,20,25,41), the expression of stationaryphase genes such as mcbA for microcin production (2) and glgCA for glycogen synthesis do not require katF (13, 38a), suggesting that additional regulators exist. We have initiated studies to identify and characterize factors that control the glycogen biosynthesis genes in E. coli. Our previous experiments showed that cyclic AMP (cAMP), cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp) stimulate the expression of the genes for the essen-

Exposure of plants to elevated temperatures results in a complexThe responses of plants to HS have received increasing attention in recent years. Elevated temperatures initiate changes in transcription and selective translation of HS mRNA encoding HSPs, thereby enhancing thermotolerance of treated plants (Nover et al., 1989; Nover, 1991;Vierling, 1991; Howarth and Ougham, 1993;Waters et al., 1996). However, the pathways by which HS signals are perceived and transduced to activate gene expression of HSPs and to induced thermotolerance are not understood.In recent years a second-messenger Ca 2ϩ was found to be involved in the perception and regulation of many responses of plants to environmental signals (Gilroy et al., 1993; Poovaiah and Reddy, 1993; Gilroy and Trewavas, 1994; Bush, 1995;Braam et al., 1996;Webb et al., 1996). [Ca 2ϩ ] cyt often shows significant changes in plant cells under the influence of various stress signals such as touch, wind stimulation, cold shock, wounding, and mechanical stimulation (Knight et al., 1991(Knight et al., , 1992(Knight et al., , 1993 Haley et al., 1995;Campbell et al., 1996; Polisensky and Braam, 1996), oxidative stress (Price et al., 1994), salinity (Lynch et al., 1989; Bush, 1996; Okazaki et al., 1996), anoxia (Subbaiah et al., 1994a; Bush, 1996;Sedbrook et al., 1996), and hypoosmotic shock (Takahashi et al., 1997). It has been suggested that a stress-induced change in [Ca 2ϩ ] cyt might be one of the primary transduction mechanisms whereby gene expression and biochemical events are altered to adapt plant cells to environmental stresses (Monroy et al., 1993;Subbaiah et al., 1994aSubbaiah et al., , 1994b Monroy and Dhindsa, 1995;Braam et al., 1996).Several authors have suggested that Ca 2ϩ -mediated second-messenger systems might be involved in the HS responses of animal cells (Lamarche et al., 1985; Calderwood et al., 1988; Landry et al., 1988; Mosser et al., 1990), although other results indicated that Ca 2ϩ was not strictly required for some HSP synthesis (Drummond et al., 1986(Drummond et al., , 1988. In plant cells Klein and Ferguson (1987) observed that the uptake of Ca 2ϩ by suspension-cultured pear cells or protoplasts was significantly enhanced during heat stress. Braam (1992) demonstrated that HS induced a strongly up-regulated expression of calmodulin-related TCH genes in cultured Arabidopsis cells, and external Ca 2ϩ was required for maximal HS induction of these TCH genes. Wu et al. (1992) also indicated that pretreatment of hypocotyl segments and etiolated seedlings of Brassica napus with the Ca 2ϩ ionophore A23187 or the Ca 2ϩ chelator EGTA to modify Ca 2ϩ homeostasis resulted in changes in the synthesis of HSPs. Using the fluorescent dye Indo-1, Biyaseheva et al. (1993) reported that HS induced a 4-fold increase in [Ca 2ϩ ] cyt in pea mesophyll protoplasts, but the further dynamic changes in [Ca 2ϩ ] cyt during HS could not be detected because of limitations in the technique.We recently described a novel technology to measure [Ca 2ϩ ] ...

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Recent work with young pigs shows that reducing dietary protein intake can improve gut function after weaning but results in inadequate provision of essential amino acids for muscle growth. Because acute administration of L-leucine stimulates protein synthesis in piglet muscle, the present study tested the hypothesis that supplementing L-leucine to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in multiple organs of post-weaning pigs. Eighteen 21-day pigs (Duroc×Landrace×Yorkshire) were fed low-protein diets (16.9% crude protein) supplemented with 0, 0.27 or 0.55% L-leucine (total leucine contents in the diets being 1.34, 1.61 or 1.88%, respectively). At 35 days of age, protein synthesis was determined using the [2H] phenylalanine flooding-dose technique. Additionally, total and phosphorylated levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) were measured in longissimus muscle and liver. Compared with the control group, dietary supplementation with 0.55% L-leucine for 2 weeks increased (P<0.05): (1) the phosphorylated levels of S6K1 and 4E-BP1; (2) protein synthesis in skeletal muscle, liver, the heart, kidney, pancreas, spleen, and stomach; and (3) daily weight gain by 61%. Dietary supplementation with 0.27% L-leucine enhanced (P<0.05) protein synthesis in proximal small intestine, kidney and pancreas. These novel findings provide a molecular basis for designing effective nutritional means to increase the efficiency of nutrient utilization for protein accretion in neonates.