Cellular utilization of cytosolic NADPH in kidney and liver cells from rats fed a normal or a vitamin D-deficient diet

The amount of reducing equivalents from NADPH generated by glucose 6-phosphate dehydrogenase activity (G6PD) used in mixed function oxidation (pathway I) or in reductive biosynthesis (pathway II) has been determined by cytochemical methods and microdensitometry in cells from the pars recta (PR) and distal convoluted tubule (DCT) of the kidney and from centrilobular (CL) and periportal (PP) hepatocytes from rats fed a normal or a vitamin D-deficient diet. In the kidney, pathway I activity was similar to that of pathway II in PR, whereas in DCT pathway II was markedly predominant. Feeding a vitamin D-deficient diet resulted in an increase in the total amount of reducing equivalents in PR and DCT. This increase was due to a rise in pathway I activity in the PR, whereas in the DCT the increase resulted from a stimulation of pathway II activity. Pathway I activity in PR was inversely correlated with plasma calcium, and was significantly decreased when calcium (1 mM) was added in vitro. In the liver the total amount of reducing equivalents generated by G6PD and both hydrogen pathways, was higher in CL than in PP hepatocytes. In CL cells, a vitamin D-deficient diet induced a significant increase in both NADPH pathways. Furthermore, in these cells pathway I activity was inversely related to plasma calcium and was significantly lowered when 1 mM calcium was added in vitro. It is concluded that vitamin D status and calcium influence the production and utilization of cytosolic reducing equivalents both in kidney and liver.     

Inhibition of IL6 in rheumatoid arthritis and juvenile idiopathic arthritis

A number of clinical trials have been done to investigate the role of interleukin-6 (IL-6) as a potential therapeutic target in rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). Most of the data testing this comes from trials of the humanized anti Il-6 receptor antibody tocilizumab. Results from clinical trials worldwide have been promising so far. Additional study will define the ultimate role of tocilizumab and Il6 inhibitors in the treatment paradigms for RA and JIA.     

Changes in insulin receptor,hexokinase and NADPH producing enzymes in Choroid plexus during experimental diabetes

The activities of insulin receptor and the enzymes hexokinase (EC 2.7.1.1) and NADP-dependent malic enzyme (EC1.1.1.40), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in rat choroid plexus in alloxan induced diabetes. A significant decrease was observed in the activities of all the enzymes except isocitrate dehydrogenase and also the choroid plexus insulin receptor activity was decreased. A reversal of the efect was observed with insulin administration to diabetic rats. It may be concluded that the enzymes of choroid plexus together with insulin receptor are directly controlled by-the concentration of insulin.     

"Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes

Protein A bound to particles of colloidal gold is widely used for cytochemical localization of antigens. A method for detecting immune complexes on "Western blots" with protein A-gold has been developed. It involved the following steps: (i) electrophoretic separation of proteins, (ii) transfer to nitrocellulose, (iii) incubation with antibodies, and (iv) staining with protein A-gold. The amount of antigen detectable was 10-50 ng. The sensitivity could be improved by subsequent photochemical silver staining (1-10 ng detected), and was then comparable to autoradiograms using iodinated protein A.     

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren’s syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.     

Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.

Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.     

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%-40% less aerial biomass and was 10%-30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp.     

Regulation of liver and brain hexose monophosphate dehydrogenases by insulin and dietary intake in the female rat

Summary Liver glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities were significantly decreased in both diabetic and fasted rats. Treatment of diabetic rats with insulin resulted in liver glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities that were significantly greater than controls. Insulin promoted an increase in food consumption that was blocked by adrenaline. Insulin, when administered together with adrenaline, restored hepatic glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenas activities of diabetic animals to control values, without altering food consumption. Brain glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities were not significantly altered by either dietary restriction, diabetes or insulin treatment. These results demonstrate a dissociation between the action of insulin on hepatic glucose 6-phosphate dehydrogenase activity and its action to increase food intake.Abbreviations NADP⁺ oxidoreductase, EC 1.1.1.49 Glucose 6-P dehydrogenase, GPD, D-glucose-6-phosphate - NADP⁺ 2-oxidoreductase (decarboxylating), EC 1.1.1.44 phosphogluconate dehydrogenase, PGD, 6-phospho-D-gluconate     

CTRP3 plays an important role in the development of collagen-induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease exhibited most commonly in joints. We found that the expression of C1qtnf3, which encodes C1q/TNF-related protein 3 (CTRP3), was highly increased in two mouse RA models with different etiology. To elucidate the pathogenic roles of CTRP3 in the development of arthritis, we generated C1qtnf3^−/− mice and examined the development of collagen-induced arthritis in these mice. We found that the incidence and severity score was higher in C1qtnf3^−/− mice compared with wild-type (WT) mice. Histopathology of the joints was also more severe in C1qtnf3^−/− mice. The levels of antibodies against type II collagen and pro-inflammatory cytokine mRNAs in C1qtnf3^−/− mice were higher than WT mice. These observations indicate that CTRP3 plays an important role in the development of autoimmune arthritis, suggesting CTRP3 as a possible medicine to treat RA.     

Effects of some drugs on the activity of glucose 6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were investigated. The enzyme was purified 2488-fold in a yield of 76.8% using ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity gel at 4°C. The drugs pental sodium, MgSO₄, vancomycin, metamizol, marcaine, and prilocaine all exhibited inhibitory effects on the enzyme. While MgSO₄ (K_i = 12.119 mM), vancomycin (K_i = 1.466 mM) and metamizol (K_i = 0.392 mM) showed competitive inhibition, pental sodium (K_i = 0.748 mM) and marcaine (K_i = 0.0446 mM) displayed noncompetitive inhibition.     

Regulation of erythrocyte membrane shape by Ca2+

In the presence of 1.0 mM ATP and MgCl₂, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca²⁺ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca²⁺ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca²⁺ (1 to 5 μM) also inhibited Mg²⁺ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca²⁺ on viscosity and phosphorylation may be due to a membrane bound Ca²⁺ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg²⁺ dependent kinase activity.     

Alterations in the activities of rat tissue hexose monophosphate dehydrogenases in response to premature weaning and dietary restriction at mid-lactation

Summary The activities of the hexose monophosphate dehydrogenases increased in adipose tissue, remained unchanged in liver and decreased in mammary gland following the weaning of rats at mid-lactation (day 14). When dietary intake was restricted at mid-lactation, the activities of the hexose monophosphate dehydrogenases increased in adipose tissue, decreased in liver, but were unaltered in mammary gland. Premature weaning on day 14 postpartum resulted in maternal increases in both plasma insulin and glucose, which peaked at day 16. The plasma insulin levels decreased from day 14 to day 18 postpartum in the normal lactating rat, and a similar trend was observed for animals on a restricted dietary intake. Daily food consumption in the lactating rat decreased from 50 g to 20 g after premature weaning. The live weight of pups raised on dams given a restricted food intake from day 14 had decreased by day 17 postpartum, whereas an increase in daily live weight gain was recorded for the litters from the lactating controls. The results demonstrate that the activities of the hexose monophosphate dehydrogenases are regulated differentially between tissues of the lactating rat.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Ultrasound power Doppler synovitis is associated with plasma IL-6 in established rheumatoid arthritis

Background and objective: Cytokines have an important role in the pathogenesis of rheumatoid arthritis (RA). Although plasma levels of IL-6 have been related to musculoskeletal ultrasound (MSUS) synovitis in early DMARD-naïve RA, there are no similar studies in established disease. Methods: 64 RA patients treated with non-biological DMARDs and 30 healthy controls were included in this prospective cross-sectional study. A blood sample was taken before evaluation of disease activity (DAS28) and ultrasonography (all tests performed in a blinded fashion). MSUS was performed by one of two ultrasound-trained rheumatologists on 10 joints of both hands. Gray scale (GS) and pD (power Doppler) synovitis were evaluated using a semi-quantitative scale (0–3) in individual joints, and their sum (score 10) was calculated. Plasma cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, TNF, IFN-γ, and VEGF) were quantified by flow cytometry. Results: Levels of all cytokines, excepting VEGF, were significantly higher in RA patients than in controls (P ⩽ 0.05). In RA patients, IL-6, but not other cytokines, correlated positively with DAS28 and swollen joint count (P ⩽ 0.01), as well as with 10-joint pD score, and GS and pD of both wrists (P < 0.01 for all tests). In multiple linear regression, the association of IL-6 with 10-joint pD score was maintained even after adjustment for DAS28. However, there was no correlation of IL-6 with tender joint count, 10-joint GS score, or presence of erosions. Conclusion: We demonstrated an association of inflammatory findings on MSUS and plasma IL-6 independently of DAS28 in established RA.     

Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis

Forty adult patients (30 women and 10 men) with rheumatoid arthritis (RA), treated with nonsteroidal anti-inflammatory drugs, were studied. Serum levels of immunoreactive ceruloplasmin, oxidase activity of the ceruloplasmin and total copper, as well as the specific oxidase activity (enzyme activity per unit of mass) and the copper/immunoreactive ceruloplasmin relationship were significantly higher in the group of patients than in the healthy control group (p < 0.001). However, no significant difference was found for the concentration of non-ceruloplasmin copper between both groups. A statistically significant negative correlation was obtained for the concentration of serum thiobarbituric acid-reacting substances with the immunoreactive ceruloplasmin and its oxidase activity in the group of patients (p < 0.005). These results suggest that in RA increases of serum copper are produced at the expense of the fraction linked to the ceruloplasmin, diminishing the proportion of apoceruloplasmin and other forms poor in copper. Although the increase in the serum concentration of ceruloplasmin might offer an additional safeguard against oxidative stress. it does not appear to have a beneficial effect upon the activity of the illness as evaluated by means the biological inflammation markers C-reactive protein, erythrocyte sedimentation rate and sialic acid.     

Paleopathologic evidence for the evolution of rheumatoid arthritis

A human skeleton recovered from a Sicilian archaeological site and dating from the Hellenistic period (330–210 B.C. ) presents a pathological pattern suggesting a transition between ankylosing spondylitis and rheumatoid arthritis, providing evidence in support of the hypothesis that rheumatoid arthritis may have recently evolved out of ankylosing spondylitis.     

The role of Toll-like receptor signalling in the pathogenesis of arthritis

Recent evidence highlighted the role of Toll-like receptors (TLRs) as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules of antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. In this review, recent studies on TLR signal transduction pathways activated by corresponding ligands are summarized and evidence for a possible role of TLR signalling in the pathogenesis of rheumatoid arthritis is discussed.     

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes

The present study demonstrates the expression of hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose-6-phosphate transporter is responsible for cortisone-cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose-6-phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.     

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.     

Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.