Search

Background: Analysis of existing mutations of Angiotensin-I-Converting Enzyme (ACE) led us to hypothesize that the carriers of damaging ACE mutations (accompanied by low ACE levels) could be at risk for the development of late-onset Alzheimer's disease (AD). Methodology/Principal findings: We quantified blood ACE levels in EDTA-containing plasma from 15 patients with 11 different heterozygous ACE mutations and estimated the effects of these mutations on ACE phenotypes, using a set of mAbs to ACE and two ACE substrates. We confirmed prior observations that the relatively frequent Y215C mutation in the N domain of ACE (present in ~1% of the population) is associated with both Alzheimer's disease (AD) and reduced plasma levels of ACE (~50% of controls), indicating that it likely results in a transport-deficient protein. In addition, we identified another 4 mutations in both ACE domains (M118T, C734Y, V992M and V997M) which are also associated with decreased ACE levels in the blood, and, thus, could be putative risk factors for late-onset AD. One of these mutations, C734Y, is likely transport-deficient, while the other mutations appear to influence ACE catalytic properties. The precipitation of mutant M118T by mAb 2D1 and ACE mutant C734Y by mAb 3F10 increased 2-3-fold compared to native ACE, and therefore, these mAbs could be markers of these mutations. Also, we identified a mutation I989T, which is associated with increased ACE levels in the blood. Conclusions/Significance: Conducting a systematic analysis of blood ACE levels in patients with ACE mutations holds promise for identifying individuals with low blood ACE levels. Such individuals may be at increased risk for late-onset AD. The patients with transport-deficient ACE mutations may benefit from therapeutic treatment with a combination of chemical and pharmacological chaperones and proteasome inhibitors, as was demonstrated previously using a cell model of the transport-deficient ACE mutation, Q1069R [Danilov et al, PLoS One, 2010]. [ABSTRACT FROM AUTHOR]

Backgrounds. Our recent analysis of 1200+ existing missense ACE mutations revealed that 400+ mutations are damaging and led us to hypothesize that carriers of heterozygous loss-of-function (LoF) ACE mutations (which result in low ACE levels) could be at risk for the development of late-onset Alzheimer's disease (AD). Methods. Here, we quantified blood ACE levels in EDTA plasma from 41 subjects with 10 different heterozygous ACE mutations, as well as 33 controls, and estimated the effect of these mutations on ACE phenotype using a set of mAbs to ACE and two ACE substrates. Results. We found that relatively frequent (~1%) AD-associated ACE mutations in the N domain of ACE, Y215C, and G325R are truly damaging and likely transport-deficient, with the ACE levels in plasma at only ~50% of controls. Another AD-associated ACE mutation, R1250Q, in the cytoplasmic tail, did not cause a decrease in ACE and likely did not affect surface ACE expression. We have also developed a method to identify patients with anti-catalytic mutations in the N domain. These mutations may result in reduced degradation of amyloid beta peptide Aβ42, an important component for amyloid deposition. Consequently, these could pose a risk factor for the development of AD. Conclusions. Therefore, a systematic analysis of blood ACE levels in patients with all ACE mutations has the potential to identify individuals at an increased risk of late-onset AD. These individuals may benefit from future preventive or therapeutic interventions involving a combination of chemical and pharmacological chaperones, as well as proteasome inhibitors, aiming to enhance ACE protein traffic. This approach has been previously demonstrated in our cell model of the transport-deficient ACE mutation Q1069R. [ABSTRACT FROM AUTHOR]

Abstract Background Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. There is a lot of data on the prevalence and genetic heterogeneity of TTV in healthy populations and in patients with various diseases now available. However, little is known about TTV load among healthy human population. In this study we analyzed TTV load in the group of 512 Russian elite athletes, who are supposed to be, by some standards, the healthiest part of the human population. Results The prevalence rate of TTV among the Russian Olympic Reserve members was 94% (for test sensitivity about 1000 genome equivalents per 1 ml of blood). Quantities varied from 103 (which corresponded to detection limit) to 1010 copies per 1 ml of blood, with median at 2.7 × 106 copies. Conclusion About 94% of healthy individuals in Russian population have more than 1000 TTV genome copies per 1 ml of blood. This result exceeds the previously published data, and can be explained by either more sensitive PCR test system or by higher TTV distribution in Russian population or both. TTV viral load neither depends on gender, nor age.

Abstract Background Freshwater planarians are widely used as models for investigation of pattern formation and studies on genetic variation in populations. Despite extensive information on the biology and genetics of planaria, the occurrence and distribution of viruses in these animals remains an unexplored area of research. Results Using a combination of Suppression Subtractive Hybridization (SSH) and Mirror Orientation Selection (MOS), we compared the genomes of two strains of freshwater planarian, Girardia tigrina. The novel extrachromosomal DNA-containing virus-like element denoted PEVE (Planarian Extrachromosomal Virus-like Element) was identified in one planarian strain. The PEVE genome (about 7.5 kb) consists of two unique regions (Ul and Us) flanked by inverted repeats. Sequence analyses reveal that PEVE comprises two helicase-like sequences in the genome, of which the first is a homolog of a circoviral replication initiator protein (Rep), and the second is similar to the papillomavirus E1 helicase domain. PEVE genome exists in at least two variant forms with different arrangements of single-stranded and double-stranded DNA stretches that correspond to the Us and Ul regions. Using PCR analysis and whole-mount in situ hybridization, we characterized PEVE distribution and expression in the planarian body. Conclusions PEVE is the first viral element identified in free-living flatworms. This element differs from all known viruses and viral elements, and comprises two potential helicases that are homologous to proteins from distant viral phyla. PEVE is unevenly distributed in the worm body, and is detected in specific parenchyma cells.

Objective. The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Methods. Using quantitative molecular-barcoded 50-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Results. Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Conclusion. Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases. [ABSTRACT FROM AUTHOR]

Focuses on crystallization and preliminary X-ray analysis of a four-copper laccase from Coriolus hirsutus. Experimental observation of the analysis; Description about crystallization processes; Structures of crystallization.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. In this protocol, differentially presented DNAs are selectively amplified using PCR. It is strongly recommended that subtractions be performed in both directions for each tester/driver DNA pair. Forward subtraction is designed to enrich for differentially presented molecules present in the tester but not in the driver; reverse subtraction is designed to enrich for differentially presented sequences present in the driver but not in the tester. Therefore, each experiment should have at least four reactions: (1) subtracted tester DNAs, (2) unsubtracted tester control, (3) reverse-subtracted tester DNAs, and (4) unsubtracted control for the reverse subtraction.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for screening a subtracted cDNA library by differential hybridization using (1) a tester-specific subtracted probe (forward-subtracted probe), (2) a driver-specific subtracted probe (reverse-subtracted probe), (3) a cDNA probe synthesized directly from tester mRNA (or tester genomic DNA), and (4) a cDNA probe synthesized directly from driver mRNA (or driver genomic DNA).

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. The major drawback of SSH is the presence of background clones that represent nondifferentially expressed DNA species in the subtracted libraries. In some cases, the number of background clones may considerably exceed the number of target clones. This protocol describes mirror orientation selection (MOS)--a simple procedure that substantially decreases the number of background clones in libraries generated by SSH. The MOS technique is based on the rationale that after PCR amplification, during SSH, background molecules will be present in one orientation only, relative to the adapter sequences. Genuine SSH clones will be present in both sequence orientations.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for subtractive hybridization using adapter-ligated tester and RsaI-digested driver DNA samples.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. We have used SSH in studies of regeneration and development on various types of model organisms (including freshwater planaria regeneration, Xenopus laevis development, and mammalian brain cortex development). We also use SSH for the analysis of strain-specific genes in bacteria with different characteristics. During these studies, a large number of differentially regulated and differentially presented genes have been identified, including transcriptional regulation factors and restriction modification enzymes. This article describes the SSH method and considerations for its use.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes a method for the use of PCR-based DNA dot blots in the differential screening of arrayed subtracted DNA clones. For high-throughput screening, a 96-well or 384-well format PCR from one of several thermal cycler manufacturers is recommended. Alternatively, single tubes can be used.

Hepatopancreas of king crab Paralithodes camtschatica produces a metalloprotease, which belongs to the astacin family, as cDNA cloning and sequencing showed. The metalloprotease has been purified chromatographically to apparent homogeneity. The purification factor was 16 and activity recovery was 20%. pH and temperature optimum have been determined. In its properties (molecular weight, pI, metal content) the metalloprotease is close to crayfish astacin. However, analysis of the enzyme sequences revealed differences, which account for differences in substrate specificities and imply a different activation mechanism.

cDNA encoding the endo-1,3-beta-d-glucanase from Spisula sachalinensis (LIV) was amplified by PCR using oligonucleotides deduced from the N-terminal end peptide sequence. Predicted enzyme structure consists of 444 amino acids with a signal sequence. The mature enzyme has 316 amino acids and its deduced amino acid sequence coincides completely with the N-terminal end (38 amino acids) of the beta-1,3-glucanase (LIV) isolated from the mollusk. The enzyme sequence from Val 121 to Met 441 reveals closest homology with Pacifastacus leniusculus lipopolysaccharide- and beta-1,3-glucan-binding protein and with coelomic cytolytic factors from Lumbricus terrestris. The mollusk glucanase also shows 36% identity and 56% similarity with beta-1,3-glucanase of the sea urchin Strongylocentrotus purpuratus. It is generally considered that invertebrate glucanase-like proteins containing the bacterial glucanase motif have evolved from an ancient beta-1,3-glucanase gene, but most of them lost their glucanase activity in the course of evolution and retained only the glucan-binding activity. A more detailed evaluation of the protein folding elicited very interesting relationships between the active site of LIV and other enzymes, which hydrolyze native glucans.

Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. In fact, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. The SSH method is based on a suppression PCR effect and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNA or genomic DNA fragments, and simplifies analysis of the subtracted library. In our hands, the SSH technique has enriched over 1000-fold for rare sequences in a single round of subtractive hybridization.