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The absorption of nitrate by field bean plants at different times of development was investigated to determine the distribution and subsequent utilization of N up to the maturity of the beans. Nodulated plants were grown in sand culture and pulse‐labelled with 15NO3 for 4 short periods during development. Whole plants were sampled at intervals until maturity and analysed for total nitrogen and 15N. There was a rapid increase in the bean dry weight and total N after fruit set and, at maturity, the beans contained about three times as much N as had ever been in the rest of the plant. Nitrate absorption continued steadily throughout plant development but nodule N fixation made a progressively greater contribution to the total N in the plant. Over a quarter of the total 15N absorbed was found in the roots immediately after each treatment and little of this was subsequently translocated upwards in spite of the large increase in shoot N taking place. Above ground the most powerful “sink” for recently absorbed and redistributed N was initially the youngest vegetative tissue. However after fruit set competition developed between the growing tip and the developing beans until at the later stages the beans became the more powerful. Quantitatively redistribution made only a small contribution to bean N. [ABSTRACT FROM AUTHOR]

A technique is described whereby active preparations of glutamic dehydrogenase (GDH) and glutamic-oxaloacetic transaminase (GOT) can be obtained from all apple tree tissues using a hand-operated coffee mill. The amount of insoluble polyvinylpyrrolidone required and the composition of the extractant have been investigated together with the degree of replication obtained and the stability of the resultant extracts. After extraction of all tissues the proportion of both GDH and GOT found in the supernatant was far greater than that in the mitochondria. Addition of calcium and some other metal ions to the assays resulted in some increase in GDH but had no effect on GOT activity. With crude extracts the effect of added calcium was small but after ultrafiltration or acid precipitation it was greatly increased. The co-factors NADH, NADPH and NAD were all active with GDH in extracts of apple leaves, stem bark and roots. No activity was found with NADP. In the presence of added calcium ions the ratios of activity NADH : NADPH and NADH : NAD were approximately 10:1 and 20:1 respectively. The seasonal variations in specific activity of GDH and GOT in apple leaves, stem bark and wood, and old and young roots were determined separately. The highest GDH activities were found in the leaves in October and in the stem bark in May, while in other tissues activities were generally higher in winter than in summer. The seasonal patterns for GOT activity were very similar to those for GDH except that in the leaves the level changed little through the year. The results are discussed in relation to published work on these enzymes in other plants and to their possible role in the apple tree. [ABSTRACT FROM AUTHOR]

Using heat-killed, formalin-stabilized Staphylococcus aureus Cowan I suspension, it was possible lo remove myeloma IgG and autoimmune anti-red blood cell antibodies from two patients' plasma following an extracorporeal procedure. The arterial anticoagulated blood was processed through a cell separator machine. The separated plasma was pumped through a bacterial filter containing S. aureus suspension. The adsorbed plasma was then reunited with the blood cells and returned to the patient through the vein. S. aureus Cowan I could selectively remove the myeloma IgG and autoimmune anti-red cell antibodies from the patients' plasma prolonging the lives of these two terminal patients. The immunoadsorption procedure appears to be a safe, practical and quick method for the removal of pathological IgG from the patients' plasma. [ABSTRACT FROM AUTHOR]

Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCbetaII is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKCbeta splicing such that PKCbetaI was downregulated and PKCbetaII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCzeta mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCbetaII inhibitor CGP53353 pointed to a role for PKCbetaII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCbetaII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCbetaII regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCbetaII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCbetaII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.

Nutrient intake during gestation has an impact on gestation parameters and subsequent lactation performance. The objectives of this experiment were to determine the impact of feeding two levels of amino acids in gestation on sow BW changes in gestation and lactation, and litter size, and to evaluate a factorial method for determining daily energy requirements. At mating, 419 sows (Camborough 15; Pig Improvement Canada, Acme, AB) were assigned randomly within Parities 1, 2 or 3+ to a gestation diet containing either 0.44% (low lysine) or 0.55% (high lysine) total lysine and 3,100 kcal DE/kg; other indispensable amino acids were adjusted to lysine based on ideal protein ratios. Feed allowance in gestation was determined factorially using estimated DE requirements for maintenance, maternal gain, and conceptus growth. Sows were allowed free access to the lactation diet. Gestation BW gain from d 0 to 110 was affected by parity (61.2, 60.0, and 42.3 kg for Parity 1, 2, and 3+, respectively; P < 0.05) but not (P > 0.10) by gestation lysine level. Sow BW changes from d 0 of lactation to weaning were affected by parity (0.5, 6.8, and 5.8 kg for Parity 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.01), but not by gestation lysine level (5.0 vs 3.8 kg for low and high lysine, respectively; P > 0.10). Total piglets born was affected by parity (11.5, 12.1, and 12.5, for Parity 1, 2, and 3+, respectively; P < 0.01) and increased with increasing sow BW gain (P < 0.05). Total piglets born alive (mean = 11.2) was increased with increasing sow BW gain (P < 0.05). Total litter weight born alive was affected by parity (15.9, 18.6, and 19.4 kg for Parities 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.05). The model used to determine daily energy intake requirements resulted in an average BW gain of 10.6 kg above the targets set by the model. Total lysine intakes greater than 10.6 g/d in gestation did not improve sow productivity. Setting target weight gains in gestation and feeding to meet these targets may not always provide predictable results due to a number of factors that affect the energy requirement in the sow.

Reproductive performance is steadily increasing within the pork industry; logically, amino acid requirements need to be redefined for sows producing larger litters. The objective of this study was to determine the threonine requirement of the high-producing lactating sow and to determine the effect of lysine on this requirement. A total of 419 PIC C-15 sows were assigned randomly to treatment within parity groups (1, 2, and 3+) and gestation treatment at d 110 of gestation. Lactation diets were formulated to contain 0.80% total lysine (tLYS) with 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, or 0.65% total threonine (tTHR) or 1.06% tLYS with 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, or 0.70% tTHR. Litters were standardized to a minimum of 11 piglets within 48 h after farrowing, and sows had free access to feed throughout lactation (lactation length = 20.1 +/- 0.1 d). Sow ADFI exceeded expectation, averaging 6.90, 7.40, and 7.20 kg/d for Parities 1, 2, and 3+, respectively. Daily tLYS intake was 58 g/d (47 g of apparent ileal digestible lysine [dLYS] per day) and 74 g/d (59 g dLYS/d) for the low- and high-lysine group, respectively. Lysine intake did not affect sow or litter performance (P > 0.10). Sows gained an average of 4.8 kg in lactation. Using regression analysis, BW gain was maximized at 0.54% tTHR for all parity groups (quadratic; P < 0.05). Litter weaning weight (67.1, 67.9, and 66.2 kg for Parities 1, 2, and 3+, respectively) and litter weight gain (2.49, 2.53, and 2.44 kg/d for Parities 1, 2, and 3+, respectively) were maximized at 0.53% tTHR using regression analysis, for all parity groups (quadratic; P < 0.05). Based on regression analysis, plasma urea nitrogen on d 10 and 18 was minimized at 0.54% tTHR (P < 0.05). Lysine levels in excess of 58 g of tLYS/d did not benefit sow or litter performance. The requirement for threonine to minimize sow tissue mobilization was 37, 40, and 38 g tTHR/d (28, 30, and 30 g of apparent ileal digestible threonine [dTHR] per day) for Parities 1, 2, and 3+ sows, respectively. The threonine required to maximize litter performance was 36, 39, and 38 g of tTHR/d (28, 30, and 29 g of dTHR/d) for Parities 1, 2, and 3+ sows, respectively. Alternatively, the requirement can be expressed as 14.3 g tTHR (11.8 g dTHR) per kilogram of litter gain.

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.

Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

Protein kinases and phosphatases are likely targets for the development of therapeutic drugs since they are involved in specific signaling pathways which regulate cell functions such as metabolism, cell cycle progression, cell adhesion, vascular function and angiogenesis. Protein phosphorylation and dephosphorylation serve as molecular switches for modulating these processes and the level and duration of each is a highly regulated process in normal cells. Several compounds that inhibit the activity of tyrosine kinases are being evaluated as cancer therapeutic agents in clinical trials. Diabetes and complications of diabetes also involve deregulated levels of protein kinases. New approaches for regulating kinase gene expression include specific antisense oligonucleotides for inhibiting post-transcriptional processing of the messenger RNA, naturally occurring products and their chemical derivatives to inhibit kinase activity, and monoclonal antibodies to inhibit receptor linked kinases. Inhibition of phosphatases also serves to alter the duration of phosphorylation by kinases. Considerations for development of effective inhibitors include non-specific actions of compounds, cellular uptake, multiple intracellular targets that can dilute the effective cellular concentration of an agent, and tissue specificity. Kinase inhibitors may allow other therapeutic agents additional time to become effective and they may act synergistically with current treatments.

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-betaII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-betaI (betaI-A10) and PKC-betaII (betaII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in betaI-A10 and betaII-A10 cells. The PKC-betaII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of betaI-A10 or betaII-A10 cells entering S phase. PKC-betaII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-betaII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-betaII, which then alters the normal inhibitory role of PKC-betaII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

Protein kinase C (PKCtheta) is a key enzyme in regulating a variety of cellular functions, including growth and differentiation. PKCtheta is the most abundant PKC isoform expressed in skeletal muscle; however, its role in differentiation and metabolism is not clear. We examined the effect of muscle cell differentiation on PKCtheta expression in human skeletal muscle cells from normal and type 2 diabetic subjects. Low levels of PKCtheta messenger RNA (mRNA) and protein were detected in human myoblasts from both types of subjects. Upon differentiation into myotubes, PKCtheta mRNA and protein were increased 12-fold in myotubes from normal subjects. In human skeletal muscle cells obtained from type 2 diabetic subjects, increases in PKCtheta mRNA and protein were not observed upon differentiation into myotubes although expression of other markers of differentiation and fusion increased. Cells from type 2 diabetic subjects also exhibited decreased insulin-stimulated glycogen synthase activity. To determine whether the up-regulation of PKCtheta was important for the metabolic actions of insulin, PKCtheta was overexpressed in L6 rat skeletal muscle cells. Increased expression of PKCtheta occurred with differentiation of skeletal muscle myoblasts to myotubes. Glycogen synthase activity was further increased in L6 myotubes stably transfected with the complementary DNA for PKCtheta. The decreased expression of PKCtheta found in cells from type 2 diabetic subjects may be linked to insulin resistance and decreased glycogen synthase activity.

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP., (Copyright 2000 Academic Press.)

Insulin regulates a diverse array of signaling pathways involved in the control of growth, differentiation, proliferation, and metabolism. Insulin increases in glucose uptake via a protein kinase C-dependent pathway in target tissues such as fat and muscle are well documented. Insulin-regulated events, however, occur in all cells. The utilization of glucose as a preferred energy source is a ubiquitous event in eukaryotic cells. In NIH-3T3 fibroblasts, insulin treatment increased levels of the cPKC and nPKC activator, diacylglycerol. Insulin-responsive 2-[(3)H]deoxyglucose uptake was stimulated in a dose-dependent manner. The overexpression of protein kinase C (PKC)betaI, -betaII, -delta, -epsilon, and -zeta was used to investigate the specificity of PKC isozymes for insulin-sensitive glucose uptake. The stable overexpression of PKCbetaII, -delta, and -epsilon resulted in increases in insulin-stimulated 2-[(3)H]deoxyglucose uptake compared to vector control cells, while basal 2-deoxyglucose uptake levels were not elevated. Overexpression of PKCbetaI and PKCzeta isozymes had no further effect on basal or insulin-stimulated 2-deoxyglucose uptake. The PKC-specific inhibitor, CGP41251, blocked insulin effects on 2-deoxyglucose uptake but not its effects on tyrosine phosphorylation of cellular substrates. Insulin-stimulated 3-O-methylglucose uptake was also greater in cells overexpressing PKCbetaII, -delta, and -epsilon, compared to control cells. The increased responsiveness was not accompanied by conversion of 3T3 cells to the adipocyte phenotype or the increased expression of insulin receptors or glucose transporters (GLUT1-type). Insulin-stimulated recruitment of GLUT1 to plasma membranes of cells overexpressing PKCbetaII, -delta, and -epsilon, was greater than that in control cells. The data suggest that more than one PKC isozyme is involved in insulin signaling pathways in fibroblasts, resulting in increased GLUT1 transporter recruitment to cell membranes., (Copyright 1999 Academic Press.)

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)-betaII in regulating MCF-7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC-betaII-specific inhibitor (CGP53353) were examined in cultures of MCF-7 human breast cancer cell line and its multidrug resistant variant (MCF-7/ADR). Cell proliferation and DNA synthesis of MCF-7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF-7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC-betaII protein and the corresponding mRNA levels were 4- to 5-fold higher in MCF-7/ADR than in MCF-7 cells. High glucose-induced decreases of PKC-betaII protein and mRNA levels were observed during the DNA synthesis phase in MCF-7 but not in MCF-7/ADR cells. Decreases in PKC-betaII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose-stimulated proliferation of MCF-7 cells. Cultures of multidrug resistant MCF-7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose-induced PKC-betaII isozyme down-regulation with concomitant acceleration of cell cycle progression in MCF-7 cells., (Copyright 1999 Wiley-Liss, Inc.)

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents., (Copyright 1999 Academic Press.)

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.