Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.     

Identification of naphthalene metabolism by white rot fungus Pleurotus eryngii

The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.     

Novel intron length polymorphic (ILP) markers from starch biosynthesis genes reveal genetic relationships in Indian wheat varieties and related species

Molecular Biology Reports - Introns experience lesser selection pressure, thus are liable for higher polymorphism. Intron Length Polymorphic (ILP) markers designed from exon-flanking introns...     

Treatment of lentil (Lens culinaris. L) seeds with various concentrations of salts to check their efficiency against seed-borne mycoflora during storage

Seed sample of lentil collected from the Swabi district were treated with NaCl and KCl at 0.01, 0.1 and 1.0% (w/w). Seed-borne mycoflora was observed at 0, 20, 40, 60 and 80 day intervals. Seed treatment with both the salts was found to be effective against storage fungi; however, KCl was more efficient against storage mycoflora such as Aspergillus species when compared with NaCl. With the passage of time, the incidence of deep-seated fungi was observed in salt-treated seed samples while untreated seed sample showed heavy infestation by the species of Aspergillus. Seed viability also remained unaffected in storage, except in seeds heavily infested with seed-borne mycoflora. Aspergillus spp. were the main cause of seed rot. Surface sterilisation of seeds with 1% Na(OCl)₂ reduced the fungal infestation of seeds. Among various concentrations of salts, 0.1% (w/w) of both salts were better in controlling seed-borne mycoflora.     

Unintended consequences of delisting routine eye exams on retinopathy screening for people with diabetes in Ontario,Canada

Background:

Routine eye examinations for healthy adults aged 20–64 years were delisted from the Ontario Health Insurance Plan in 2004, but they continue to be insured for people with diabetes regardless of age. We sought to assess whether the delisting of routine eye examinations for healthy adults had the unintended consequence of decreasing retinopathy screening for adults with diabetes.

Methods:

We used administrative data to calculate eye examinations for people with diabetes ages 40–64 years and 65 years and older in each 2-year period from 1998 to 2010. We examined differences by sex, income, rurality and type of health care provider. We used segmented linear regression to assess the change in trend before and after 2004.

Results:

For people with diabetes aged 65 years and older, eye examinations rose gradually from 1998 to 2010, with no substantial change between 2004 and 2006. For people with diabetes aged 40–65 years, there was an 8.7% (95% confidence interval [CI] 6.3%–11.1%) decrease in eye examinations between 2004 and 2006. Results were similar for all population subgroups. Ophthalmologic examinations decreased steadily for both age groups during the study period, and there was a decline in optometry examinations for people ages 40–65 years after 2004.

Interpretation:

The delisting of routine eye examinations for healthy adults in Ontario had the unintended consequence of reducing publicly funded retinopathy screening for people with diabetes. More research is needed to understand whether patients are being charged for an insured service or to what degree misunderstanding has prevented patients from seeking care.Diabetic retinopathy is the leading cause of new cases of blindness in people of working age.¹ In the United States, about 40% of adults with diabetes aged 40 years and older have retinopathy, and 8% have vision-threatening retinopathy.² Studies suggest that, if untreated, 50% of patients with proliferative diabetic retinopathy become legally blind within 5 years, compared with only 5% of patients who receive early treatment.³ Regular dilated eye examinations are effective for early detection and monitoring of asymptomatic retinopathy in people with diabetes⁴ and are recommended by clinical practice guidelines.^5,6In Ontario, Canada’s most populous province, medically necessary services are covered by the Ontario Health Insurance Plan (OHIP) for all permanent residents and Canadian citizens living in the province.⁷ Under OHIP, routine eye examinations were fully insured for all children and adults until November 1, 2004. At that time, routine eye examinations ceased being insured for healthy adults aged 20–64 years, but continued to be insured for children aged 19 years and younger and for adults aged 65 years and older.⁸ Regardless of age, adults with diabetes and some other medical conditions affecting the eye, as well as adults receiving social assistance, continued to have an annual eye examination covered by OHIP. Insured examinations are at no cost to the patient and are reimbursed to the provider at about Can$40. In contrast, healthy adults aged 20–64 years are required to pay out-of-pocket or through private insurance for a routine eye examination, with fees set at the discretion of the optometrist⁹ or physician.¹⁰Health policy experts suggest that delisting services from insurance schemes can have unpredictable effects.¹¹ Understanding the effect of delisting on care is particularly important as governments face fiscal pressures and contemplate further reductions in what is publicly insured.¹² We sought to assess whether delisting routine eye examinations for healthy middle-aged adults in Ontario had the unintended consequence of decreasing retinopathy screening for middle-aged adults with diabetes, even though eye examinations continued to be insured for this population.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.     

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.