Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation

BACKGROUND: The peptidyl prolyl cis/trans isomerase (PPIase) Parvulin (Par14/PIN4) is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro. RESULTS: In this study we confirm by RT-PCR the existence of a longer Parvulin isoform expressed in all tissues examined so far. This isoform contains a 5' extension including a 75 bp extended open reading frame with two coupled SNPs leading to amino acid substitutions Q16R and R18S. About 1% of all Parvulin mRNAs include the novel extension as quantified by real-time PCR. The human Parvulin promoter is TATA-less and situated in a CpG island typical for house keeping genes. Thus, different Parvulin mRNAs seem to arise by alternative transcription initiation. N-terminally extended Parvulin is protected from rapid proteinaseK degradation. In HeLa and HepG2 cell lysates two protein species of about 17 and 28 KDa are detected by an antibody against an epitope within the N-terminal extension. These two bands are also recognized by an antibody towards the PPIase domain of Parvulin. The longer Parvulin protein is encoded by the human genome but absent from rodent, bovine and non-mammalian genomes. CONCLUSION: Due to its molecular weight of 16.6 KDa we denote the novel Parvulin isoform as Par17 following the E. coli Par10 and human Par14 nomenclature. The N-terminal elongation of Par17-QR and Par17-RS suggests these isoforms to perform divergent functions within the eukaryotic cell than the well characterized Par14

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

<p>Abstract</p> <p>Background</p> <p>The parvulin-type peptidyl prolyl <it>cis/trans </it>isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study.</p> <p>Results</p> <p>Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions.</p> <p>Conclusion</p> <p>Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae.</p

Experimental study of the role of physicochemical surface processing on the IN ability of mineral dust particles

During the measurement campaign FROST 2 (FReezing Of duST 2), the Leipzig Aerosol Cloud Interaction Simulator (LACIS) was used to investigate the influence of various surface modifications on the ice nucleating ability of Arizona Test Dust (ATD) particles in the immersion freezing mode. The dust particles were exposed to sulfuric acid vapor, to water vapor with and without the addition of ammonia gas, and heat using a thermodenuder operating at 250 °C. Size selected, quasi monodisperse particles with a mobility diameter of 300 nm were fed into LACIS and droplets grew on these particles such that each droplet contained a single particle. Temperature dependent frozen fractions of these droplets were determined in a temperature range between −40 °C ≤T≤−28 °C. The pure ATD particles nucleated ice over a broad temperature range with their freezing behavior being separated into two freezing branches characterized through different slopes in the frozen fraction vs. temperature curves. Coating the ATD particles with sulfuric acid resulted in the particles' IN potential significantly decreasing in the first freezing branch (T>−35 °C) and a slight increase in the second branch (T≤−35 °C). The addition of water vapor after the sulfuric acid coating caused the disappearance of the first freezing branch and a strong reduction of the IN ability in the second freezing branch. The presence of ammonia gas during water vapor exposure had a negligible effect on the particles' IN ability compared to the effect of water vapor. Heating in the thermodenuder led to a decreased IN ability of the sulfuric acid coated particles for both branches but the additional heat did not or only slightly change the IN ability of the pure ATD and the water vapor exposed sulfuric acid coated particles. In other words, the combination of both sulfuric acid and water vapor being present is a main cause for the ice active surface features of the ATD particles being destroyed. A possible explanation could be the chemical transformation of ice active metal silicates to metal sulfates. The strongly enhanced reaction between sulfuric acid and dust in the presence of water vapor and the resulting significant reductions in IN potential are of importance for atmospheric ice cloud formation. Our findings suggest that the IN concentration can decrease by up to one order of magnitude for the conditions investigated

Corrigendum to "Experimental study of the role of physicochemical surface processing on the IN ability of mineral dust particles" published in Atmos. Chem. Phys., 11, 11131–11144, 2011

In the abstract of the manuscript, the following sentence needs to be corrected: “A possible explanation could be the chemical transformation of ice active metal silicates to metal sulfates.” “metal silicates” is wrong. It should be “aluminosilicates”

Immersion Freezing of Kaolinite: Scaling with Particle Surface Area

This study presents an analysis showing that the freezing probability of kaolinite particles from Fluka scales exponentially with particle surface area for different atmospherically relevant particle sizes. Immersion freezing experiments were performed at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). Size-selected kaolinite particles with mobility diameters of 300, 700, and 1000 nm were analyzed with one particle per droplet. First, it is demonstrated that immersion freezing is independent of the droplet volume. Using the mobility analyzer technique for size selection involves the presence of multiply charged particles in the quasi-monodisperse aerosol, which are larger than singly charged particles. The fractions of these were determined using cloud droplet activation measurements. The development of a multiple charge correction method has proven to be essential for deriving ice fractions and other quantities for measurements in which the here-applied method of size selection is used. When accounting for multiply charged particles (electric charge itself does not matter), both a time-independent and a time-dependent description of the freezing process can reproduce the measurements over the range of examined particle sizes. Hence, either a temperature-dependent surface site density or a single contact angle distribution was sufficient to parameterize the freezing behavior. From a comparison with earlier studies using kaolinite samples from the same provider, it is concluded that the neglect of multiply charged particles and, to a lesser extent, the effect of time can cause a significant overestimation of the ice nucleation site density of one order of magnitude, which translates into a temperature bias of 5–6 K

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>sequence two additional in-frame ATG codons (highlighted in blue). The only other 5' ATG codon is present in the sequence; it is out of frame and followed by a TAA stop codon (both underlined). All in-frame stop codons are marked in red. Only the human sequence displays an extended open reading frame, both as QR and RS isoform (SNPs highlighted in grey)

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>n mRNA contains a 92 bp extension at the 5' side depicted in white. Primers used for RT-PCR are indicated by arrows. Primers 246 and 247 are complementary to the sequences around the two start ATG codons, 248 binds at the sequence around the TAA stop codon. . RT-PCR products with mRNA from liver, kidney and Caco-2 cells after 30 amplification cycles. Primers 246 and 248 yield a 488 bp PCR product only on those Par14 mRNAs with 5' extension. Primers 247 and 248 give rise to a 385 bp DNA fragment with all Par14 mRNAs. All longer RT-PCR products were eluted from the gel and sequenced. . Two start ATG codons are indicated in bold capital letters. The sequence of Parvulin common to both originally described cDNAs by Uchida . [GenBank:] [3] and Rulten . [GenBank:] [9] begins at the caa codon depicted in bold. The peptide sequence used for antibody production is shaded in grey. Two SNPs are shown leading to amino acid substitutions Q16R and R18S

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>. Reaction products were separated by 17.5% SDS-PAGE following autoradiography. . Equal amounts of 35S-methionine labeled Par17-QR, Par17-RS and Par14 were incubated with increasing concentrations of proteinaseK (PK) for 30 min at 10°C. Enzyme concentrations are given in μg/ml. Reactions were stopped by an excess of PMSF, separated by SDS-PAGE and subjected to autoradiography. . Western blot of HepG2 (lane 1 and 3) and HeLa (lane 2 and 4) cell lysates. Proteins were separated by SDS-PAGE with MES as running buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes. Blots were incubated with pre-immune (lane 1 and 2) or anti-Par17 serum (lane 3 and 4; both at 500-fold dilution). . Coding sequences for Par17-RS and -QR were subcloned in the pET-28 vector with N-terminal His6 tag and expressed in . Lysates were subjected to reducing SDS-PAGE in MES buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes and incubated with the anti-Par17 antibody. -, before IPTG induction; RS, Par17-RS lysates; QR, Par17-QR lysates. Coomassie stained gel of lysates to show equal loading. Par17 fusions with His6 tag and thrombin cleavage sites show apparent molecular weights of about 22 KDa in SDS-PAGE. The induction band in E. coli lysates and the corresponding band recognized by Ab-EXT are labeled with arrows. The lower migrating band may be caused by proteolytic degradation. . Par14 coding sequence was expressed as GFP fusion in HeLa cells (lane 3). Lane 1 and 2 are HeLa cell lysates not transfected with this construct. Lysates were subjected to reducing SDS-PAGE in Tris-glycine buffer with MagicMark as protein standard, transferred to nitrocellulose membranes and incubated with anti-Par17 and anti-GFP antibodies. Coomassie stained gel of lysates to show equal loading