Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)     

Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His?Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered?myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.     

Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans

Although glyphosate-resistant (GR) technology is used in most countries producing soybeans (Glycine max L.), there are no particular fertilize recommendations for use of this technology, and not much has been reported on the influence of glyphosate on GR soybean nutrient status. An evaluation of different cultivar maturity groups on different soil types, revealed a significant decrease in macro and micronutrients in leaf tissues, and in photosynthetic parameters (chlorophyll, photosynthetic rate, transpiration and stomatal conductance) with glyphosate use (single or sequential application). Irrespective of glyphosate applications, concentrations of shoot macro- and micronutrients were found lower in the near-isogenic GR-cultivars compared to their respective non-GR parental lines Shoot and root dry biomass were reduced by glyphosate with all GR cultivars evaluated in both soils. The lower biomass in GR soybeans compared to their isogenic normal lines probably represents additive effects from the decreased photosynthetic parameters as well as lower availability of nutrients in tissues of the glyphosate treated plants.     

Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting

During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P₂ analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P₂ localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P₂-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.     

Urinary Excretion of Zinc and Metabolic Control of Patients with Diabetes Type 2

The objective of this study was to assess urinary excretion of zinc and evaluation parameters of metabolic control in type 2 diabetic patients. Thirty-one type 2 diabetic patients, of both genders, with 5.8 ± 5.6 years average time of the disease, age range 20–60 years, were selected. Evaluation of the nutritional status was performed using anthropometric measurements. To evaluate food consumption, the 3-day alimentary log method was used, and its analysis was performed using a software. Determination of urinary zinc was by atomic absorption spectrophotometry. From the obtained results, it was concluded that 51.6% of the patients were overweight. The mean of found waist circumference was 100.4 and 92.2 cm for men and women, respectively. Blood glucose and glycated hemoglobin values were higher than reference values, and plasma albumin concentration was adequate. The median of found urinary zinc excretion was 474.9 μg/24 h, within normal standards (300–600 μg/day). Regarding diet composition, calorie and protein concentration were above recommendation, while mean zinc concentration was adequate. This data allow the conclusion that the evaluated patients presented adequate urinary zinc excretion in comparison with reference values.     

Point mutations in the HIV-1 matrix protein turn off the myristyl switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.