A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

University of California, Santa Cruz

A total number of 100 patients were examined over a period of one year to find out any correlation between diabetic maculopathy and diabetic nephropathy. Twenty-two belonged to insulin dependent diabetes mellitus group and 78 patients were in the non-insulin dependent diabetes mellitus group who were suffering from diabetes over a period of 0-25 years and more. Another group comprising 6 patients were chosen from these 100 patients who were suffering from nephropathy diagnosed clinically and on pathological investigations. Background and proliferative--both types of diabetic retinopathy were found with increased incidence with the persistence of the disease in both types of diabetes mellitus. Nine patients had diabetic maculopathy, out of which 6 patients (66.66%) were suffering from diabetic nephropathy. Proliferative diabetic retinopathy is more common in insulin dependent diabetes mellitus than in non-insulin dependent diabetes mellitus of more than 25 years of duration. Maculopathy is more common in non-insulin dependent diabetes mellitus and there is strong correlation between diabetic maculopathy and diabetic nephropathy.

Association of diabetic maculopathy with diabetic nephropathy--a study.

To study the ocular abnormalities in cases of vitiligo, 100 patients were examined who underwent a series of investigations e.g., complete haemogram, urine analysis, blood biochemistry and chest x-ray and 70 cases were taken as control. Patients with systemic diseases such as diabetes mellitus, thyroid and auto-immune disorders were excluded. Detailed ocular examinations including recording of vision, slit-lamp biomicroscopy, gonioscopy, ophthalmoscopy and examination of fundus by 90 D lens were done. Of 100 vitiligo patients, 23% showed hypopigmented sports on the iris, 18% pigmentation on anterior chamber, 9% retinal pigment epithelium hypopigmentation, 5% uveitis, 11% had chorioretinal degeneration and 34% had no ocular findings. There lies a strong association of ocular disorder in patients with vitiligo.

Clinical pattern of ocular manifestations in vitiligo.

A 65-year-old male patient presented with a rapidly increasing mass in his left eye. It did not show any intra-ocular extension as examined by slit lamp, gonioscopy, indirect ophthalmoscopy and ultrasonography, nor there was any sign of systemic metastasis. The mass was excised completely. Buccal mucous membrane grafting was done to cover up the lower forniceal area and conjunctival autograft taken from right eye and placed over upper fornix and bulbar area. Postoperative radiotherapy was suggested. Histopathological examination of the mass revealed the diagnosis of malignant melanoma of conjunctiva.

Malignant melanoma of conjunctiva--a case report.

Selective dsDNA-templated formation of copper nanoparticles in solution.

The autocatalytic photochemical reaction, which is potentially controlled by any selected nucleic acid, is highly sequence specific and not inhibited by its products, was developed. This reaction generates colored and fluorescent products, which can be monitored by the naked eye.

An autocatalytic chromogenic and fluorogenic photochemical reaction controlled by nucleic acids.

We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging.

Hybridization and reaction-based fluorogenic nucleic acid probes.

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N',N'-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2'-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD4 and conjugates of oligonucleotides with N,N,N',N'-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N',N'-tetramethylrhodamine using green light.

To study the effects of anti-oxidant on tear film parameters in patients with diabetes mellitus, a total of 100 patients of diabetes mellitus with defective Schirmer test and tear film break-up time were enrolled in this study and they were given vitamin C (1000 mg/day) and vitamin E (400 IU/day) for 30 days. Schirmer test and tear break-up, time were re-examined after 1 month. There was significant improvement in values for Schirmer test (p < 0.001) and tear break-up time. Oxidative stress has an influence on how diabetes mellitus affects various systems of body. This study clearly depicted im- portant roles of vitamins C and E in improving the well-being of the ocular surface.

Effect of anti-oxidant on tear film in patients suffering from diabetes mellitus.

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads were conducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations show common origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliation of different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity of Pseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selection pressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community among different agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value (D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-borne pathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population of plant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp.

The amyloid beta peptide 42 (Aβ42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer's disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the β-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aβ aggregates in neurotoxicity.

Introduction of d-Glutamate at a Critical Residue of Aβ42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to β-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high β-sheet characteristics. Our approach can be extended to other residues of Aβ, as well as further amyloidogenic peptides.

Subrata Dutta

Department of Chemistry

University of California, Santa Cruz

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Subrata Dutta

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry

University of California, Santa Cruz

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Department of Chemistry