Synthetic peptides derived from the C-terminal 6 kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development

Background

Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site.

Methods

In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential.

Results

The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5.

Conclusions

Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress.

General significance

These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.     

Update meta-analysis on MMP-7 − 181A>G polymorphism and cancer risk: Evidence from 25 studies

Background

The matrix metalloproteinase (MMP) can degrade various components of the extracellular matrix and its functional genetic polymorphism may be associated with cancer development. The common MMP-7 (− 181A>G) genetic polymorphism has been reported to be functional and may contribute to genetic susceptibility to cancers. However, the association between MMP-7 (− 181A>G) and cancer risk remains inconclusive.

Methods

To better understand the role of MMP-7 (− 181A>G) polymorphism in global cancer, we conducted this comprehensive meta-analysis encompassing 6392 cases and 7665 controls.

Results

Overall, the MMP-7 (− 181A>G) polymorphism was associated with higher cancer risk. In the stratified analyses, significant associations were found between the MMP-7 (− 181A>G) polymorphism and gastric cancer, ESCC and gynecologic cancer. We also observed that the GG genotype might modulate colorectal cancer risk comparing with the AA genotype (OR = 1.31[1.02–1.69]). Moreover, a significantly increased cancer risk was found among Asian populations. When stratified by study design, significantly elevated susceptibility to cancer was found among population-based studies.

Conclusions

These findings suggested that the MMP-7 (− 181A>G) genetic polymorphism may contribute to the susceptibility of cancers, especially among Asian population.     

MMP-1 promoter genotype and haplotype association with posterior tibial tendinopathy

Purpose

Posterior tibial tendon (PTT) is particularly vulnerable and its insufficiency is recognized as the main cause of adult acquired flatfoot. Some patients have a predisposition without clinically recognized cause, suggesting that individual characteristics play an important role in tendinopathy. The objective of the present study is to investigate the association of − 519 (rs1144393) matrix metalloproteinase-1 (MMP-1) polymorphism and the − 1607 (rs1799750) and − 519 MMP-1 haplotypes and risk of PTT dysfunction.

Methods

The test group included 50 females who presented PTT dysfunction Grade 2 or 3, and who were submitted to surgical treatment, with histopathological examination of the tendon and magnetic resonance image (MRI) confirming tendinopathy, while the control group was 100 asymptomatic women who present intact PTT at MRI. We analyzed functional polymorphisms MMP-1 and their haplotypes using polymerase chain reaction and restriction fragment length analysis.

Results

There was a significant difference in the presence of the different alleles and genotypes between the control group and test group for the MMP-1 gene (p ≤ 0.01). The G allele of the − 519 MMP-1 polymorphism increased susceptibility to degeneration in the PTT tendon and seems to be a genetic risk factor. Global haplotype analysis indicated a significant difference between both groups (p < 0.0001). Haplotypes G–2G and A–2G had statistically significant risk effect on PTT insufficiency. G–2G, p < 0.001; OR = 5.72 (CI, 2.84–11.52) and A–2G p = 0.002, OR = 3.95 (CI, 1.65–9.44).

Conclusion

According to our results, − 519 MMP-1 isolated and − 1607/− 519 MMP-1 haplotypes are associated to tendinopathy in posterior tibial tendon.     

Impact of MMP-3 and TIMP-3 gene polymorphisms on prostate cancer susceptibility in North Indian cohort

Purpose

Matrix metalloproteinases (MMPs) have been implicated in progression and metastases of different tumors. The balance between the MMPs and their natural inhibitors (tissue inhibitors of matrix metalloproteinases; TIMP) seem to be an important factor related to its role. The purpose of this study was to evaluate polymorphisms in the MMP-3 and TIMP-3 genes for their associations with prostate cancer (PCa) risk in North Indians.

Materials and methods

Genotypes were determined by PCR-RFLP (Polymerase Chain Reaction Restriction Fragment Length Polymorphism) method in 150 PCa patients and 200 age matched controls of similar ethnicity.

Results

We found significant association in the MMP-3(1171)5A/6A and TIMP-3 (1298) C/T polymorphism with PCa risk. Variant genotype (5A/5A) of MMP-3(1171)5A/6A polymorphism had a high PCa risk (p = 0.037, OR = 3.52, 95%CI = 1.08–11.5). Individuals with TIMP-3 (1298) CT genotype as well as T allele showed reduced risk of PCa (p < 0.001; OR = 0.31; 95%CI = 0.18–0.52, and p = 0.001; OR = 0.49; 95%CI = 0.32–0.75). This effect was even more evident in case of T allele carrier (CT + TT) (p < 0.001; OR = 0.36; 95%CI = 0.22–0.59). Overall no significant association was observed statistically in MMP-3 and TIMP-3 with any of the grading stages and smoking habits in PCa. Haplotype analysis of MMP-3 showed that A-5A-A was associated with three folds (OR = 3.06; 95%CI = 1.71–5.47; p < 0.001) increased risk in PCa patients.

Conclusion

This is the first reported association between polymorphisms in the MMP-3 and TIMP-3 gene and PCa risk and supports the hypothesis that the protease/antiprotease balance has an important role. Due to the small sample size further investigations need to be done to prove a statistical significant correlation between the MMP/TIMP expression and clinicopathological parameters.     

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors

Background

Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs.

Methods

Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching.

Results

Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC₅₀ values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC₅₀ values are far above the concentrations needed to activate or inhibit the caspases.

Conclusion

The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs.

General significance

Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

The polymorphism − 1131T > C in apolipoprotein A5 gene is associated with dyslipidemia in Brazilian subjects

Background

Polymorphisms in apolipoprotein A5 gene (APOA5) have been associated with higher triglyceride levels in many populations. The aim of the study was to determine the allelic and genotypic distribution of the APOA5 − 1131T > C polymorphism and to identify the association of the genetic variant and the risk for dyslipidemia.

Methods

We genotyped 109 dyslipidemic subjects and 107 controls. The total cholesterol, triglycerides and HDL-c were determined enzymatically. Comparison of means among groups was calculated by ANOVA. Significant differences among groups were evaluated by Student–Newman–Keuls test.

Results

The minor allele C was more frequent in dyslipidemic subjects than controls (p = 0.019) and confers an increased individual risk for dyslipidemia (OR = 1.726, CI 95% = 1.095–2.721). The genotype analysis by gender showed that this allele was more frequent in dyslipidemic males (p = 0.037; OR = 2.050, CI 95% = 1.042–4.023). When participants were analyzed according to genotypes TT and TC/CC, C-carriers presented higher cholesterol and triglycerides levels than TT homozygous (p = 0.046 and 0.049, respectively).

Conclusions

The allele C confers higher total cholesterol and triglycerides levels in dyslipidemic adults. The APOA5 − 1131T > C polymorphism is associated with dyslipidemia in male subjects.     

Early senescence in heterozygous ABCA1 mutation skin fibroblasts: A gene dosage effect beyond HDL deficiency?

Purpose

Homozygous ABCA1 gene mutation causes Tangier disease (TD). The effects reported in heterozygous state regard plasma HDL, cell cholesterol efflux and coronary artery disease. We investigated whether in vitro replicative skin fibroblast senescence shown in TD proband (Hom), his father (Het), and in a healthy control might be induced in a “gene-dosage way”.

Methods

Senescence was evaluated by staining test for β-Galactosidase and telomere length (TL) on fibroblast DNA at different replicative stages. ABCG1 and LDLR (low density lipoprotein receptor) gene expression was also evaluated.

Results

Hom cells showed early senescent morphology and reduced growth at all passages in vitro. The cell positive percentage for β-Galactosidase test was highly increased in Hom compared to Het cells at late replicative status (66.1% vs 41.3% respectively). TL was significantly shorter at high stage either in Hom (p < 0.0001) or in Het (p < 0.005). At early replication cycles ABCG1 gene expression was about 3-fold higher in Hom compared to Het cells (0.44 vs 0.14 arbitrary unit).

Conclusions

ABCA1 gene mutation may have “gene-dosage way” effect on in vitro fibroblast senescence. Furthermore, increased ABCG1 and LDLR gene expression could highlight a role of ABCA1 on cytoskeleton regulation associated to cell cholesterol metabolism.     

Protective effect of melatonin against transient global cerebral ischemia-induced neuronal cell damage via inhibition of matrix metalloproteinase-9

Aims

Melatonin possesses various pharmacological effects including neuroprotective effects against brain ischemia. Post-ischemic increases in matrix metalloproteinase-9 (MMP-9) expression and activity mainly contribute to neuronal damage by degradation of the extracellular matrix. This study was designed to examine whether melatonin has a neuroprotective effect and an influence on MMP-9 in transient global brain ischemia.

Main methods

Mice were subjected to 20 min of global brain ischemia and sacrificed 72 h later. Melatonin (30 mg/kg) was administered 30 min before and 2 h after ischemia as well as once daily until sacrifice.

Key findings

Hippocampal pyramidal cell damage after ischemia was significantly decreased by melatonin. As observed by zymography, melatonin inhibited the increase of MMP-9 activity after ischemia. In the brain sections, the increased gelatinase activity was mainly observed in the hippocampus after ischemia and melatonin also reduced gelatinase activity. The laminin and NeuN expression levels were reduced in the hippocampal CA1 and CA2 regions after ischemia, and melatonin reduced laminin degradation and neuronal loss. A TUNEL assay demonstrated that there were TUNEL-positive cells in the hippocampus and the number of TUNEL-positive cells was significantly decreased by melatonin. There was no difference in the ischemia-induced hippocampal neuronal damage between the vehicle- and melatonin-treated groups of MMP-9 knock-out mice.

Significance

These data demonstrate that melatonin suppressed the occurrence of neuronal injury, which might be partly due to its inhibitory effects on MMP-9 in addition to its anti-oxidative effects. MMP-9 may be an important key target of melatonin in neuroprotection against global ischemia.     

Tracing the pathway of compositional changes in bone mineral with age: Preliminary study of bioapatite aging in hypermineralized dolphin's bulla

Background

Studies of mineral compositional effects during bone aging are complicated by the presence of collagen.

Methods

Hypermineralized bullae of Atlantic bottlenose dolphins of < 3 months, 2.5 years, and 20 years underwent micrometer-scale point analysis by Raman spectroscopy and electron microprobe in addition to bulk analysis for carbon.

Results

Bulla central areas have a mineral content of ~ 96 wt.% and 9–10 wt.% carbonate in their bioapatite, which is ~ 2 wt.% more than edge areas. Ca/P atomic ratios (~ 1.8) and concentrations of Mg, S, and other minor/trace elements are almost constant in central areas over time. Maturity brings greater over-all homogeneity in mineral content, stoichiometry, and morphology throughout the central and edge areas of the bullae. During aging, edge areas become less porous, whereas the concentration of organics in the edge is reduced. Enhancement of coupled substitutions of CO₃^2 − for PO₄^3 − and Na for Ca during aging increases carbonate content up to ~ 10 wt.% in the adult bulla.

Conclusions

1) Changes in physical properties during aging did not occur simultaneously with changes in chemical properties of the bone mineral. 2) Compositional changes in bone mineral were minor during the neonatal to sub-adult stage, but significant during later maturity. 3) Na and CO₃ concentrations co-vary in a 1:1 molar proportion during aging. 4) The mineral's crystallinity did not decrease as CO₃ concentration increased during aging.

General significance

Hypermineralized dolphin's bulla, due to extreme depletion in collagen, is an ideal material for investigating mineralogical changes in bioapatite during bone aging.     

Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine

Background

Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III₁ upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition.

Methods

Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine.

Results

Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~ 10⁵ M^− 1 for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy–entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines.

Conclusion

These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes.

General significance

It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines

Background

There is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients.

Methods

Galectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state.

Results

Galectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80 ~ 10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by ~ 90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth.

Conclusions

Among the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact.

General significance

The study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.     

Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment

Background

Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment.

Methods

Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150 K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting.

Results

Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~ 120 fs to ~ 3 ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors.

General significance

The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.     

Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity

Background

Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation.

Methods

Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy.

Results

In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment.

Conclusions

Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells.

General Significance

Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.     

Genetic variants in selenoprotein P plasma 1 gene (SEPP1) are associated with fasting insulin and first phase insulin response in Hispanics

Context

Insulin resistance is not fully explained on a molecular level, though several genes and proteins have been tied to this defect. Knockdowns of the SEPP1 gene, which encodes the selenoprotein P (SeP) protein, have been shown to increase insulin sensitivity in mice. SeP is a liver-derived plasma protein and a major supplier of selenium, which is a proposed insulin mimetic and antidiabetic agent.

Objective

SEPP1 single nucleotide polymorphisms (SNPs) were selected for analysis with glucometabolic measures.

Participants and measures

The study included1424 Hispanics from families in the Insulin Resistance Atherosclerosis Family Study (IRASFS). Additionally, the multi-ethnic Insulin Resistance Atherosclerosis Study was used. A frequently sampled intravenous glucose tolerance test was used to obtain precise measures of acute insulin response (AIR) and the insulin sensitivity index (S_I).

Design

21 SEPP1 SNPs (tagging SNPs (n = 12) from HapMap, 4 coding variants and 6 SNPs in the promoter region) were genotyped and analyzed for association.

Results

Two highly correlated (r² = 1) SNPs showed association with AIR (rs28919926; Cys368Arg; p = 0.0028 and rs146125471; Ile293Met; p = 0.0026) while rs16872779 (intronic) was associated with fasting insulin levels (p = 0.0097). In the smaller IRAS Hispanic cohort, few of the associations seen in the IRASFS were replicated, but meta-analysis of IRASFS and all 3 IRAS cohorts (N = 2446) supported association of rs28919926 and rs146125471 with AIR (p = 0.013 and 0.0047, respectively) as well as rs7579 with S_I (p = 0.047).

Conclusions

Overall, these results in a human sample are consistent with the literature suggesting a role for SEPP1 in insulin resistance.     

IDH1 mutations is prognostic marker for primary glioblastoma multiforme but MGMT hypermethylation is not prognostic for primary glioblastoma multiforme

Purpose

To establish the frequency of IDH1 mutations and MGMT methylation in primary glioblastomas.

Experimental design

We screened primary glioblastoma multiforme (GBM) in a population-based study for IDH1 mutations and MGMT methylation and correlated them with clinical data.

Results

IDH1 mutations were detected in 5 of 40 primary glioblastomas (12,5%). Primary GBM patients carrying IDH1 mutations were significantly younger, mean age of 41 ± 5.06 years, than patients with wild-type IDH1, mean age of 57 ± 2,29 years, p = 0.011. The mean survival time of all GBM patients with and without IDH1 mutations was 19 months (5 cases) and 16 months (35 cases), respectively (p > 0,05). MGMT methylation was detected in 13 of the 40 patients (32,5%). MGMT-promoter methylation did not correlate with overall survival (OS; p > 0,05).

Conclusion

In summary, our study is the first study to investigate the IDH1 mutation status and MGMT methylation in primary GBMs in Turkish population and confirmed IDH1 mutation as a genetic marker for also primary GBMs. Our data are still insufficient for definite ascertainment; and our preliminary results suggest: IDH1 status shows an association with younger age and there is a lack of association between IDH1 mutation and survival time. Furthermore MGMT promoter methylation had no prognostic value and lower frequency in primary glioblastomas.     

Polymorphisms of folate metabolism-related genes and survival of patients with colorectal cancer in the Korean population

Background

5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for colorectal cancer (CRC), and the major targets of 5-FU are thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), and reduced folate carrier 1 (RFC1). We hypothesized that polymorphisms in the genes encoding these proteins would be associated with CRC patient survival.

Patients and methods

We genotyped the following polymorphisms in 372 CRC patients: TS enhancer region (TSER), TS 1494del6, MTHFR 677C > T and 1298A > C, and RFC1 − 43T > C, 80G > A, and 696C > T. Using Kaplan–Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between these polymorphisms and overall survival (OS).

Results

The combined TS 1494 0bp6bp + 6bp6bp genotype was associated with reduced OS compared to the TS 1494 0bp0bp genotype. Among rectal cancer patients, the RFC1 − 43CC and 80AA genotypes were associated with favorable OS.

Conclusions

Our data suggest that TS and RFC1 polymorphisms are associated with CRC prognosis in Korean patients. Further studies are needed to verify these findings.     

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 +.