Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10–30 s) localized alkalinization of the apoplast after a lag period of 10–20 s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20 min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100 μm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50 mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca^2 + channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca^2 +-channel blockers, such as La^3 +, Gd^3 +, and Zn^2 +. The maximal pH values attained at the incision site (~ 10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.     

Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice: Implications for impaired mitochondrial biogenesis and synaptic damage

The purpose of our study was to assess mitochondrial biogenesis and distribution in murine primary neurons. Using 5-bromo-2-deoxyuridine (BrdU) incorporation and primary neurons, we studied the mitochondrial biogenesis and mitochondrial distribution in hippocampal neurons from amyloid beta precursor protein (AβPP) transgenic mice and wild-type (WT) neurons treated with oxidative stressors, rotenone and H₂O₂. We found that after 20 h of labeling, BrdU incorporation was specific to porin-positive mitochondria. The proportion of mitochondrial area labeled with BrdU was 40.3 ± 6.3% at 20 h. The number of mitochondria with newly synthesized DNA was higher in AβPP neuronal cell bodies than in the cell bodies of WT neurons (AβPP, 45.23 ± 2.67 BrdU-positive/cell body; WT, 32.92 ± 2.49 BrdU-positive/cell body; p = 0.005). In neurites, the number of BrdU-positive mitochondria decreased in AβPP cultures compared to WT neurons (AβPP, 0.105 ± 0.008 BrdU-positive/μm neurite; WT, 0.220 ± 0.036 BrdU-positive/μm neurite; p = 0.010). Further, BrdU in the cell body increased when neurons were treated with low doses of H₂O₂ (49.6 ± 2.7 BrdU-positive/cell body, p = 0.0002 compared to untreated cells), while the neurites showed decreased BrdU staining (0.122 ± 0.010 BrdU-positive/μm neurite, p = 0.005 compared to the untreated). BrdU labeling was increased in the cell body under rotenone treatment. Additionally, under rotenone treatment, the content of BrdU labeling decreased in neurites. These findings suggest that Aβ and mitochondrial toxins enhance mitochondrial fragmentation in the cell body, and may cause impaired axonal transport of mitochondria leading to synaptic degeneration.     

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [¹⁴C]oleic acid (OA) and [¹⁴C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO₂, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.     

ACE I/D and MTHFR C677T polymorphisms are significantly associated with type 2 diabetes in Arab ethnicity: A meta-analysis

In this meta-analysis study, SNPs were investigated for their association with type 2 diabetes (T2D) in both Arab and Caucasian ethnicities. A total of 55 SNPs were analyzed, of which 11 fulfilled the selection criteria, and were used for analysis. It was found that TCF7L2 rs7903146 was significantly associated with a pooled OR of 1.155 (95%C.I. = 1.059–1.259), p < 0.0001 and I² = 78.30% among the Arab population, whereas among Caucasians, the pooled OR was 1.45 (95%C.I. = 1.386–1.516), p < 0.0001 and I² = 77.20%. KCNJ11 rs5219 was significantly associated in both the populations with a pooled OR of 1.176(1.092–1.268), p < 0.0001 and I² = 32.40% in Caucasians and a pooled OR of 1.28(1.111–1.475), p = 0.001 among Arabs. The ACE I/D polymorphism was found to be significantly associated with a pooled OR of 1.992 (95%C.I. = 1.774–2.236), p < 0.0001 and I² = 83.20% among the Arab population, whereas among Caucasians, the pooled OR was 1.078 (95%C.I. = 0.993–1.17), p = 0.073 and I² = 0%. Similarly, MTHFR C677T polymorphism was also found to be significantly associated among Arabs with a pooled OR of 1.924 (95%C.I. = 1.606–2.304), p < 0.0001 and I² = 27.20%, whereas among Caucasians, the pooled OR was 0.986 (95%C.I. = 0.868–1.122), p = 0.835 and I² = 0%. Meanwhile PPARG-2 Pro12Ala, CDKN2A/2B rs10811661, IGF2BP2 rs4402960, HHEX rs7923837, CDKAL1 rs7754840, EXT2 rs1113132 and SLC30A8 rs13266634 were found to have no significant association with T2D among Arabs. In conclusion, it seems from this study that both Arabs and Caucasians have different SNPs associated with T2D. Moreover, this study sheds light on the profound necessity for further investigations addressing the question of the genetic components of T2D in Arabs.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.     

The relationship between five widely-evaluated variants in CDKN2A/B and CDKAL1 genes and the risk of type 2 diabetes: A meta-analysis

The genes encoding two cyclin-dependent kinases-inhibitor-2A/B (CDKN2A/B) and 5 regulatory subunit-associated protein-like 1 (CDKAL1) have been investigated extensively in associations with type 2 diabetes; the results, however, are often irreproducible. We therefore sought to evaluate these associations by performing a meta-analysis on five widely-evaluated variants from the two genes. There were 38 studies (patients/controls: 51,940/52,234) for rs10811661, 16 studies (20,029/24,419) for rs564398 in CDKN2A/B gene, and 27 studies (28,383/47,635) for rs7756992, 26 studies (28,816/31,713) for rs7754840, 21 studies (29,260/38,400) for rs10946398 in CDKAL1 gene. Overall risk estimates for type 2 diabetes conferred by rs10811661-T, rs564398-A, rs7754840-C, rs7756992-G, and rs10946398-C alleles were 1.17 (95% CI: 1.10–1.23; P < 0.0005; I² = 83.9%), 1.1 (95% CI: 1.0–1.21; P = 0.051; I² = 88.3%), 1.24 (95% CI: 1.18–1.3; P < 0.0005; I² = 74.3%), 1.2 (95% CI: 1.11–1.3; P < 0.0005; I² = 92.0%), and 1.19 (95% CI: 1.1–1.29; P < 0.0005; I² = 90.8%), respectively. There was evident publication bias for rs564398 and rs7754840. Subgroup analyses by ethnicity showed remarkable divergences in risk estimate for rs564398 between Asians (odds ratio [OR] = 1.01; 95% CI: 0.86–1.19; P = 0.868) and Caucasians (OR = 1.19; 95% CI: 1.03–1.35; P = 0.012) (P < 0.05). For all variants examined, the results of studies in retrospective design or with population-based controls were comparative with that of overall studies. In meta-regression analyses, age was found to exert a significant influence on the association between rs10811661 and type 2 diabetes (P = 0.003), as well as between rs7754840 and gender (P = 0.034). Taken together, our findings provide evidence for a significant contribution of CDKN2A/B gene rs10811661 and CDKAL1 gene rs7756992 and rs10946398 to type 2 diabetes.     

The RTK/ERK pathway is associated with prostate cancer risk on the SNP level: A pooled analysis of 41 sets of data from case–control studies

Prostate cancer (PCa) is a malignant disease influencing numerous men worldwide every year. However, the exact pathogenesis and the genes, environment, and other factors involved have not been explained clearly. Some studies have proposed that cell signaling pathways might play a key role in the development and progression of PCa. According to our previous study, the RTK/ERK pathway containing nearly 40 genes was associated with PCa risk. On the basis of these genes, we conducted a meta-analysis with our own Chinese Consortium for Prostate Cancer Genetics (ChinaPCa) study and available studies in the databases to describe the association between the pathway and PCa on the SNP level. The results suggested that rs4764695/IGF1 (recessive model: pooled OR = 0.92, 95%CI = 0.852–0.994, P = 0.034; I² = 0%, P = 0.042; allele analysis: pooled OR = 0.915, 95%CI = 0.874–0.958, P = 0; I² = 0%, P = 0.424; codominant model: OR = 0.835, 95%CI = 0.762–0.916, P = 0; I² = 0%, P = 0.684) and rs1570360/VEGF (recessive model: OR = 0.596, 95%CI = 0.421–0.843, P = 0.003; I² = 23.9%, P = 0.269; codominant model: OR = 0.576, 95%CI = 0.404–0.820, P = 0.002; I² = 49.1%, P = 0.140) were significantly associated with PCa. In subgroup analysis, the relationship was also found in Caucasians for IGF1 (dominant model: OR = 0.834, 95%CI = 0.769–0.904, P = 0; allele analysis: OR = 0.908, 95%CI = 0.863–0.955, P = 0; AA vs CC: OR = 0.829, 95%CI = 0.750–0.916, P = 0; AC vs CC: OR = 0.837, 95%CI = 0.768–0.912, P = 0). In addition, in Asians (allele analysis: OR = 0.21, 95%CI = 0.168–0.262, P = 0) and Caucasians (recessive model: OR = 0.453, 95%CI: 0.240–0.855, P = 0.015; codominant model: OR = 0.464, 95%CI = 0.240–0.898, P = 0.023) for VEGF, the association was significant. The results indicated that rs4764695/IGF1 and rs1570360/VEGF might play a key role in the development and progression of PCa. On the SNP level, we suggest that the study gives us a new view of gene-pathway analysis and targeted therapy for PCa.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum–mitochondria coupling

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)–mitochondria communication, as it allows for a more efficient transfer of Ca²⁺ into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER–mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3 h of GLP-1 treatment, paralleled by increased Ca²⁺ transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca²⁺ increases in GLP-1 treated cells. Inhibiting both Ca²⁺ release from the ER and Ca²⁺ entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER–mitochondria communication in VSMC, resulting in higher mitochondrial activity.     

Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca Regulation and 14-3-3 Binding

Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca^2 + binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca^2 + binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (K_D = 0.9 μM) and present the model of a double-headed Myo1c–14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.     

Characterization of novel store-operated calcium entry effectors

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca^2 + entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1–5 μM and inhibition at > 30 μM. Unfortunately, it is also able to modify the activity of other Ca^2 + transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation. Previously, we have shown that SOCE potentiation by 2-APB depends on the presence of the central boron-oxygen core (BOC) and that the phenyl groups determine the sensitivity of the molecule to inhibit and/or potentiate the SOCE.     

Effects of apoplastic sucrose on carbohydrate pools and sucrose efflux of mesophyll protoplasts of pea (Pisum sativum)

The effects of the apoplastic, i.e. external, concentration of sucrose (0–30 m M ) on O₂ evolution, O₂ consumption, starch, sucrose, glucose and fructose content, and uptake and efflux of sucrose in mesophyll protoplasts of Pisum sativum L. cv. Fenomen were studied. Neither photosynthesis, dark respiration, sucrose nor starch content change with increased apoplastic sucrose concentration. The contents of glucose and fructose in the protoplasts increase with increased apoplastic sucrose concentration. The sucrose efflux increases with increased external sucrose concentrations between 1 and 5 m M , but above this range the efflux decreases with increased external sucrose concentrations. These findings indicate that although external sucrose does not enter the protoplasts, there is a relationship between the external sucrose pool and the internal pools of sugars in the mesophyll protoplasts. The results suggest an active sucrose efflux from the protoplasts at physiological concentrations of apoplastic sucrose (max 5 m M ) and a simple diffusion mechanism at higher concentrations.     

Meta-analysis on association between the ATP-binding cassette transporter A1 gene (ABCA1) and Alzheimer's disease

Purpose

In the past decade, a number of case–control studies have been carried out to investigate the relationship between ABCA1 polymorphisms and Alzheimer's disease (AD). However, these studies have yielded contradictory results. To investigate this inconsistency, a meta-analysis was performed.

Methods

Databases including PubMed, Web of Science, EMBASE and CNKI were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

A total of 13 case–control studies, involving 6214 patients and 6034 controls for ABCA1 polymorphisms were included. In a combined analysis, the summary per-allele odds ratio for AD of the 219 K was 1.03 (95% CI: 0.93–1.14, p = 0.56). A meta-analysis of studies on the 883 M and 1587 K variant showed no significant overall association with AD, yielding a per-allele odds ratio of 1.10 (95% CI: 0.96–1.26, p = 0.16), and 1.09 (95% CI: 0.97–1.24, p = 0.16) respectively. Similar results were also found for heterozygous and homozygous. In the subgroup analysis by ethnicity, sample size, APOE status and onset type, no significant associations were found in almost all genetic models.

Conclusions

In summary, there was no significant association detected between ABCA1 R219K, I883M and R1587K polymorphisms and risk for AD.     

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF–OA was prepared and its activity and structural changes were compared with α-LA–OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF–OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF–OA was obtained by heat-treatment at pH 8.0 with LD₅₀ of 4.88, 4.95 and 4.62 μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA–OA. Similar to HAMLET, LF–OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF–OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein–OA complexes.     

Association of ANRIL polymorphism (rs1333049:C>G) with myocardial infarction and its pharmacogenomic role in hypercholesterolemia

Single nucleotide polymorphisms (SNPs) of non-coding RNA in the INK4 locus (ANRIL) have been found to be associated with myocardial infarction (MI). However, the effect of rs1333049:C>G in INK4 locus in familial hypercholesterolemia patients and on lipid profile of the patients has not been studied in Pakistan. We therefore investigated the association of SNP rs1333049:C>G with MI as well as familial hypercholesterolemia patients and also determined the effect of genotype on lipid levels in a northern Pakistani population. A case–control association study was performed in which 611 individuals (294 patients, 290 healthy controls and 27 patients from hypercholesterolemia families) were genotyped for rs1333049:C>G, using an Allele specific polymerase chain reaction. We found a significant association of rs1333049:C>G with MI (χ² = 22.3, p < 0.001). The frequency of risk genotype CC was significantly different from the healthy controls (p < 0.001, χ² = 22.3). The risk allele C was at a higher frequency in the MI patients as compared to the controls (odds ratio [OR] = 1.55 (95% confidence interval [CI] = 1.22–1.96), p < 0.001). The logistic regression analysis for the genotype distribution resulted in strong association of risk allele C with MI under recessive model (OR = 3.17 (95% CI = 1.85–5.44) p < 0.001). When the data were further analyzed along the lines of gender, a significant association with both males and females was observed.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Association of adiponectin (AdipoQ) and sulphonylurea receptor (ABCC8) gene polymorphisms with Type 2 Diabetes in North Indian population of Punjab

In Type 2 Diabetes (T2D), adiponectin (AdipoQ) and sulphonylurea receptor genes (ABCC8) are important targets for candidate gene association studies. The single nucleotide polymorphisms (SNPs) in these genes have been associated with features of the metabolic syndrome across various populations. The present case–control study undertaken in the population of Punjab, evaluates the association of + 45T>G polymorphism in AdipoQ gene; and Exon16-3C>T as well as Exon18C>T polymorphisms in ABCC8 gene with T2D. These SNPs were genotyped in 200 T2D cases and 200 non-diabetic healthy controls using the PCR-RFLP method. The frequency of the minor G-allele for AdipoQ+ 45(T>G) polymorphism was significantly higher in T2D cases (29.0%) than in controls (21.5%) [P = 0.02, OR = 1.49 (1.07–2.04)]. The genetic model analysis revealed that the G-allele cumulatively provides nearly 1.59–1.78 fold increased risk to T2D under the additive (P = 0.009; OR = 1.59, 1.12–2.25 at 95% CI), dominant (TG/GG vs. TT) (P = 0.034, OR = 1.64, 1.04–2.56 at 95% CI) and codominant model (TG vs. TT/GG) (P = 0.014; OR = 1.78, 1.12–2.82 at 95% CI) after adjusting for confounding factors. However, no difference in the distribution of genotype and allele frequencies was observed for both the ABCC8 polymorphisms. The distribution of obesity profiles (BMI, WC and WHR) was also significantly different between cases and controls (P < 0.05). Higher BMI and central obesity were observed to increase the risk of T2D. G-allele of + 45(T>G) polymorphism in the adiponectin gene appears to be associated with increased risk of T2D, but the polymorphisms in sulphonylurea receptor gene do not seem to be associated with T2D in the population of Punjab.