Dehydrin stress proteins in birch buds in regions with contrasting climate

Dehydrin stress proteins were identified in buds of silver birch (Betula pendula Roth) grown in regions with contrasting climate, Karelia and Central Yakutia, using specific antibodies. Two types of dehydrins present in the plant buds, proteins with average (56–73 kDa) and low (14–21 kDa) molecular weight, as well as 17-kDa dehydrin, were detected in all studied plants. The most sensitive to seasonal changes are 14- to 21-kDa dehydrins, the level of which, regardless of the region where the birch grows, significantly increased during the autumn–winter period. The intraspecific polymorphism of dehydrins was more pronounced in B. pendula grown under the sharply continental climate of Yakutia, which is probably due to the peculiarities of the adaptation of woody plants to the extremely low temperatures of the cryolitic zone.     

Intracellular Salmonella metabolism

Growth of Salmonella inside infected host cells is a key aspect of their ability to cause local enteritis or systemic disease. This growth depends on exploitation of host nutrients through a large Salmonella metabolism network with hundreds of metabolites and enzymes. Studies in cell culture infection models are unravelling more and more of the underlying molecular and cellular mechanisms but also show striking Salmonella metabolic plasticity depending on host cell line and experimental conditions. In vivo studies have revealed a qualitatively diverse, but quantitatively poor, host‐Salmonella nutritional interface, which on one side makes Salmonella fitness largely resilient against metabolic perturbations, but on the other side severely limits Salmonella biomass generation and growth rates. This review discusses goals and techniques for studying Salmonella intracellular metabolism, summarises main results and implications, and proposes key issues that could be addressed in future studies.     

Kinetic model of the gating system of the sodium channel in a Ranvier node membrane

A kinetic model of the sodium channel gating system consisting of four subunits with three states--closed (X), open (Y) and inactivated (Z)--is proposed. For the channel to conduct, all the four subunits must be in the open state. The transitions between states X and Y are independent, while those between states X and Z are coupled, so that for the particle considered transition of one of two neighbouring particles into state Z increases the activation energy of the step by kT. The model fits rather well to the experimental data.     

Molecular evidence for a type C retrovirus etiology of the lymphoproliferative disease of turkeys.

Recently, we isolated from the blood of lymphoproliferative disease (LPD)-affected turkeys a type C retrovirus distinct from the avian leukosis-sarcoma virus complex and the reticuloendotheliosis virus group. We present molecular evidence for the implication of this virus in the LPD of turkeys. Using complementary DNA of LPD viral RNA, we found that the LPD viral genome is specifically and efficiently transcribed (2,500 copies per cell) in LPD tumor cells. Moreover, the LPD tumor cells contained newly inserted LPD viral information (5 to 10 copies per haploid genome), which was not present before the infection. From the absence of LPD virus-specific sequences in the normal cell genome of turkeys, it was concluded that the LPD virus is not an endogenous virus of turkeys. DNA-DNA annealing experiments revealed that the degree of sequence homology between LPD viral complementary DNA and cellular DNA of turkeys was not higher than that between LPD viral complementary DNA and cellular DNA of other species, thus indicating that the virus does not originate from turkeys.     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.     

Identification of a family of animal sphingomyelin synthases

Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.     

Reconstitution of EBV latent but not lytic antigen-specific CD4+ and CD8+ T cells after HIV treatment with highly active antiretroviral therapy

The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation. Despite this, EBV load remained unaltered, even after 5 years of therapy, although a decline in both CD4+ and CD8+ T cells specific for the lytic EBV protein BZLF1 suggested a decreased EBV reactivation rate. In contrast, latent EBV Ag EBNA1-specific CD4+ and CD8+ T cell responses were restored after 5 years of treatment to levels comparable to healthy individuals. In two individuals who were treated by HAART late during HIV progression, a lymphoma developed shortly after initiation of HAART, despite restoration of EBV-specific CD4+ and CD8+ T cells. In conclusion, long-term HAART does not alter the EBV DNA load, but does lead to a restoration of EBNA1-specific T cell responses, which might allow better control of EBV-infected cells when applied early enough during HIV infection.     

Toward positional cloning of the curly tail gene

BACKGROUND: The curly tail (ct) mutant mouse is one of the best-studied mouse models of spina bifida. The ct mutation has been localized to distal chromosome 4 in two independent studies and was recently postulated to be in the Grhl-3 gene. METHODS: A recombinant BALB/c-ct strain was generated and used to precisely map the ct gene. RESULTS: We report the absence of gross chromosomal abnormalities and the precise mapping of the ct gene to a 3-Mb region at 135 Mb (66 cM) from the centromere, closely linked to the polymorphic microsatellite marker D4Mit148. Candidate genes, Idb3, Wnt4, Cdc42, and perlecan, all localized in the critical region, were studied by sequence and expression analyses. Our data indicate that these genes in all probability do not account for the ct phenotype. In addition, our expression data do not provide strong evidence that Grhl-3 is indeed the ct gene. CONCLUSIONS: The ct gene has not yet been identified. A total of 29 candidate genes remain present in the critical region. Refined mapping studies need to be performed to further narrow the region and additional candidate genes need to be examined. Supplementary material for this article can be found on the Birth Defects Research (Part A) website (http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2005/73/tables_S3-S6.doc).     

The plasma peptidome

Background

It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.

Methods

A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.

Results

Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~?14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥?E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~?26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~?0.001 or less. Log₁₀ peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.

Conclusions

The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log₁₀ intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.