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Quantification of dolichol in the human lens with different types of cataracts.
Get PDFPURPOSE:
To quantify and characterize dolichol species in cataractous and clear human lenses.
METHODS:
Whole lenses were collected from cadaver eyeballs from the C.H. Nagri Eye Bank and Red Cross Society Eye Bank (Ahmedabad, India). Cataractous nuclei were collected after extracapsular cataract extraction (ECCE). Wet weight for all the lenses was taken and were stored at -50 degrees C until used. Dolichol was extracted using a standard protocol and then analyzed using High Performance Liquid Chromatography (HPLC) on a 4.6 mmx60 mm Hypersil-Octadecylsilane (ODS; 3 microm) reversed phase column using a Waters dual pump apparatus, a Waters gradient programmer, and an ultraviolet (UV) detector set at 210 nm. Dolichol 13 was used as an internal standard, and dolichol mixture from the liver was used as an external qualitative standard.
RESULTS:
The highest dolichol concentration was found in nuclear cataract (2.54+/-0.6 microg) followed by posterior subcapsular cataract (1.4+/-0.35 microg), and the lowest levels were observed in cortical cataract (0.37+/-0.06 microg). The level of dolichol concentration in cataractous lenses was statistically significantly higher than the levels in clear lenses (1.0+/-04.3 microg; p<0.01).
CONCLUSIONS:
The dolichol concentration was significantly higher in lenses with nuclear cataract. A significant difference in dolichol concentration was observed between the different types of cataract. It suggests that dolichol and other isoprenoids may be associated with cataractogenesis
A novel method for screening the glutathione transferase inhibitors
Get PDFBACKGROUND: Glutathione transferases (GSTs) belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST) inhibitors, a novel method was designed.
RESULTS: Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV) and F2 peptide (KWRNKKFELGLEFPNL), can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB) and glutathione as substrates) was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction.
CONCLUSION: It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes
Bacteriophage-encoded functions engaged in initiation of homologous recombination events
Get PDFRecombination plays significant role in bacteriophage biology. Functions promoting recombination are involved in key stages of phage multiplication and drive phage evolution. Their biological role is reflected in the great variety of phages existing in the environment. This work presents the intricacy of recombination of DNA phages and highlights the discrete character of phage-encoded recombination functions (anti-RecBCD activity, 5’ 3’ DNA exonucleases, homologous pairing proteins, etc.). Specifically accented are phage-mediated events which initiate genetic exchange. Importance of recombination is reviewed based on the accepted coli-phage T4 and models, the recombination system of phage P22, and the recently characterized recombination functions of phages SPP1 and Che9c. Key steps of the molecular mechanisms involving phage recombination functions and their application in molecular engineering are discussed
Recombinant Mouse GM-CSF is glycosylated in transgenic tobacco and maintains its biological activity
No full textThe granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine with many important applications
and, due to its immunostimulatory properties, could also be used as a vaccine adjuvant. A simple strategy
to produce recombinant mouse GM-CSF (mGM-CSF) in transgenic Nicotiana tabacum plants was used in this
study. The mGM-CSF cDNA followed by the sequence encoding endoplasmic reticulum retention signal (KDEL)
was cloned into the ImpactVector under the control of the strong promoter from the gene encoding a small subunit
of Rubisco. In transgenic plants the accumulation level of recombinant mGM-CSF varied in the individual
transformants from 8 to 19 μg/g of fresh leaf tissue, which makes up to 0.3% of total soluble protein. In most
analyzed plants, the apparent molecular weight of the recombinant protein was larger than predicted due to its
N-glycosylation, presumably in 2 sites. The recombinant plant-produced murine GM-CSF retained its biological
activity as confi rmed in vitro in proliferation assay using a mouse cell line, which is growth-dependent on
GM-CSF
Mutagenic potency of MMS-induced 1meA/3meC lesions in E. coli.
Get PDFThe mutagenic activity of MMS in E. coli depends on the susceptibility of DNA bases to methylation and their repair by cellular defense systems. Among the lesions in methylated DNA is 1meA/3meC, which is recently recognized as being mutagenic. In this report, special attention is focused on the mutagenic properties of 1meA/3meC which, by the activity of AlkB-dioxygenase, are quickly and efficiently converted to natural A/C bases in the DNA of E. coli alkB(+) strains, preventing 1meA/3meC-induced mutations. We have found that in the absence of AlkB-mediated repair, MMS treatment results in an increased frequency of four types of base substitutions: GC-->CG, GC-->TA, AT-->CG, and AT-->TA, whereas overproduction of PolV in CC101-106 alkB(-)/pRW134 strains leads to a markedly elevated level of GC-->TA, GC-->CG, and AT-->TA transversions. It has been observed that in the case of AB1157 alkB(-) strains, the MMS-induced and 1meA/3meC-dependent argE3-->Arg(+) reversion occurs efficiently, whereas lacZ(-)--> Lac(+) reversion in a set of CC101-106 alkB(-) strains occurs with much lower frequency. We considered several reasons for this discrepancy, namely, the possible variance in the level of the PolV activity, the effect of the PolIV contents that is higher in CC101-106 than in AB1157 strains and the different genetic cell backgrounds in CC101-106 alkB(-) and AB1157 alkB(-) strains, respectively. We postulate that the difference in the number of targets undergoing mutation and different reactivity of MMS with ssDNA and dsDNA are responsible for the high (argE3-->Arg(+)) and low (lacZ(-) --> Lac(+)) frequency of MMS-induced mutations
The Role of Annexin 1 in Drought Stress in Arabidopsis
No full textAnnexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were upregulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of
intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca2+ affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses
Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin.
No full textHuman serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB)>multi-metal binding site (MBS)>N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9+/-5, 1.1+/-0.5, and 0.9+/-0.3x10(4)M(-1), respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed
Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae.
Get PDFMost of the prokaryotic and eukaryotic replicative polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells
Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System
Get PDFIn humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and
type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature
deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme
of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes
involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be
readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely
devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the
effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of
simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of
sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the
mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal
microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation
in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid
pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response
compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol,
mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the
level of glycerophospholipids
Adenovirus dodecahedron, as a drug delivery vector.
Get PDFStability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs