Dietary protein level interacts with ω-3 polyunsaturated fatty acid deficiency to induce hypertension

Background : Dietary ω-3 fatty acid deficiency can lead to hypertension in later life; however, hypertension is affected by numerous other dietary factors. We examined the effect of altering the dietary protein level on blood pressure in animals deficient or sufficient in ω-3 fatty acids.

Methods : Female rats were placed on one of four experimental diets 1 week prior to mating. Diets were either deficient (10% safflower oil; DEF) or sufficient (7% safflower oil, 3% flaxseed oil; SUF) in ω-3 fatty acids and contained 20 or 30% casein (DEF20, SUF20, DEF30, SUF30). Offspring were maintained on the maternal diet for the duration of the experiment. At 12, 18, 24, and 30 weeks, blood pressure was assessed by tail cuff plethysmography.

Results : At both 12 and 18 weeks of age, no differences in blood pressure were observed based on diet, however, by 24 weeks hypertension was evident in DEF30 animals; there were no blood pressure differences between the other groups. This hypertension in DEF30 group was increased at 30 weeks, with systolic, diastolic, and mean arterial pressure all elevated.

Conclusions : These results indicate that the hypertension previously attributed to ω-3 fatty acid deficiency is dependent on additional dietary factors, including protein content. Furthermore, this study is the first to plot the establishment of ω-3 fatty acid deficiency hypertension over time.

Alteration of copper physiology in mice overexpressing the human menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).

Decreased fatty acit Â-oxidation in riboflavin-responsive, multiple acylocoenzyme a dehydrogenase-deficient patients is associated with an increase in uncoupling protein-3

Riboflavin-responsive, multiple acylcoenzyme A dehydrogenase deficiency (RR-MAD), a lipid storage myopathy, is characterized by, among others, a decrease in fatty acid (FA) ß-oxidation capacity. Muscle uncoupling protein 3 (UCP3) is up-regulated under conditions that either increase the levels of circulating free FA and/or decrease FA ß-oxidation. Using a relatively large cohort of seven RR-MAD patients, we aimed to better characterize the metabolic disturbances of this disease and to explore the possibility that it might increase UCP3 expression. A battery of biochemical and molecular tests were performed, which demonstrated decreases in FA ß-oxidation and in the activities of respiratory chain complexes I and II. These metabolic alterations were associated with increases of 3.1- and 1.7-fold in UCP3 mRNA and protein expression, respectively. All parameters were restored to control values after riboflavin treatment. We postulate that the up-regulation of UCP3 in RR-MAD is due to the accumulation of muscle FA/acylCoA. RR-MAD is an optimal model to support the hypothesis that UCP3 is involved in the outward translocation of an excess of FA from the mitochondria and to show that, in humans, the effects of FA on UCP3 expression are direct and independent of fatty acid ß-oxidation.

Protein kinase C isozymes as potential therapeutic targets in immune disorders

Background: Members of the protein kinase C (PKC) family are key signalling mediators in immune responses, and pharmacological inhibition of PKCs may be useful for treating immune-mediated diseases. Objective: To review and discuss the insights gained so far into various PKC isozymes and the therapeutic potential and challenges of developing PKC inhibitors for immune disorder therapy. Methods: A literature review of the role of PKCs in immune cell signalling and recent studies describing immune functions associated with PKC isozyme deficiency in relevant mouse disease models, followed by specific case studies of current and potential therapeutic strategies targeting PKCs. Results/conclusion: There is vast amount of data supporting PKC isozymes as attractive drug targets for certain immune disorders. Although the development of specific PKC isozyme inhibitors has been challenging, some progress has been made. It remains to be seen if broad-scale or isozyme-selective inhibition of PKC will have clinical efficacy.

Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin-dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86-fold) and CaMKKβ (10.1 ± 2.59-fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK-1 also increased 10-fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl22/2 mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl22/2 mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl22/2 mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2 mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity associated metabolic dysfunction.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Deficiency in apoptosis-inducing factor recapitulates chronic kidney disease via aberrant mitochondrial homeostasis

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.

A question mark on zinc deficiency in 185 million people in Pakistan- possible way out

This paper reviews research published in recent years concerning the effects of zinc deficiency, its consequences, and possible solutions. Zinc is an essential trace element necessary for over 300 zinc metalloenzymes and required for normal nucleic acid, protein, and membrane metabolism. Zinc deficiency is one of the ten biggest factors contributing to burden of disease in developing countries. Populations in South Asia, South East Asia, and sub-Saharan Africa are at greatest risk of zinc deficiency. Zinc intakes are inadequate for about a third of the population and stunting affects 40% of preschool children. In Pakistan, zinc deficiency is an emerging health problem as about 20.6% children are found in the levels of zinc, below 60 μg/dL. Signs and symptoms caused by zinc deficiency are poor appetite, weight loss, and poor growth in childhood, delayed healing of wounds, taste abnormalities, and mental lethargy. As body stores of zinc decline, these symptoms worsen and are accompanied by diarrhea, recurrent infection, and dermatitis. Daily zinc requirements for an adult are 12-16 mg/day. Iron, calcium and phytates inhibit the absorption of zinc therefore simultaneous administration should not be prescribed. Zinc deficiency and its effects are well known but the ways it can help in treatment of different diseases is yet to be discovered. Improving zinc intakes through dietary improvements is a complex task that requires considerable time and effort. The use of zinc supplements, dietary modification, and fortifying foods with zinc are the best techniques to combat its deficiency.

Reduced glutathione biosynthesis in Drosophila melanogaster causes neuronal defects linked to copper deficiency.

Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.

New insights into CNS requirements for the copper-ATPase, ATP7A. Focus on "Autonomous requirements of the Menkes disease protein in the nervous system".

COPPER IS INDISPENSABLE for development and function of the central nervous system (CNS). This is dramatically illustrated by the severe neuropathological deficits in Menkes disease, an X-linked copper deficiency disorder resulting from mutation of the gene that encodes an essential copper transporting P1B-type ATPase, ATP7A. Since its discovery over two decades ago, the role of ATP7A in copper transport and homeostasis has been inextricably linked to satisfying systemic and CNS requirements for copper. In a recent issue of American Journal of Physiology-Cell Physiology, Hodgkinson et al. (8) describe an important body of work, which for the first time distinguishes the CNS requirement for ATP7A from the CNS requirement for copper.