The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Histological assessment of cellular half-life in tissues in vivo

The assessment of cellular half-life is of fundamental importance for cell biology and biomedicine. Here, we show that cellular half-life in tissues can be histologically measured under steady state conditions in vivo by analyzing the loss of 5-bromo-2′-deoxyuridine (BrdU)-labeled cells over time after withdrawal of long-term BrdU labeling. To achieve efficient continuous cell labeling, we implanted BrdU-containing subcutaneous slow-release pellets into 12-month-old male Fischer 344 rats, delivering BrdU at a dose of 75 mg/kg per day over 1 (n = 20) or 3 weeks (n = 20). Four to five rats each were killed directly after the labeling or 1, 3, and 7 weeks post-labeling. Cellular half-life after withdrawal of BrdU was analyzed by nonlinear regression analysis of the labeling index, using a model of one-phase exponential decay. We initially validated our technique in the duodenum, where we determined a half-life of 2.4 days for crypt cells. Next, we applied this method to other tissues, and found a half-life of 2.2 weeks for cardiac endothelial cells, and of 5–6 days for pancreatic duct cells. In conclusion, we believe that this novel approach is an important step forward in the histological assessment of cellular half-life.     

Identification of progesterone and 5α‐dihydroprogesterone (5α‐DHP) in zebras by two‐dimensional high‐performance liquid chromatography and their dependence on the state of reproduction

Because of its low levels in late pregnancy, the relationship of progesterone to pregnancy maintenance in Equidae is not obvious. This study investigated the levels of progesterone (4‐pregnane‐3,20‐dione; P4) and 5α‐dihydroprogesterone (5α‐DHP) during pregnancy in zebras in relation to reproductive state. Blood samples from female zebras (Equus burchelli, E. zebra hartmannae, E. grevyi) were taken at Dvur Kralove Zoo. Progesterone and 5α‐DHP were separated by high‐performance liquid chromatography techniques and detected by cross‐reacting antibodies. Identification of progestins was achieved by comparing the identity of peaks of the samples with a standard. In E. z. hartmannae progesterone, values reached 50 ng/mL at the beginning of pregnancy and dropped to levels below 1 ng/mL during the second half of pregnancy. In contrast, 5α‐DHP increased up to 123 and 183 ng/mL during late pregnancy in E. z. hartmannae and E. burchelli, respectively. In E. grevyi, 5α‐DHP levels of 368 ng/mL were obtained during pregnancy, whereas progesterone values were similar in pregnant and non‐pregnant individuals. These marked differences in the course of progesterone and 5α‐DHP levels point to the importance of 5α‐DHP for pregnancy maintenance in zebras. Zoo Biol 18:325–333, 1999. © 1999 Wiley‐Liss, Inc.     

No response of plasma substance P, but delayed increase of interleukin-1 receptor antagonist to acute psychosocial stress

Psychosocial stress has been shown to induce inflammatory reactions, followed by the release of immunosuppressive glucocorticoids. This may be mediated by catecholamines or other stress reactive substances such as neuropeptides or cytokines. We here set out to explore the effects of acute psychosocial stress on plasma levels of substance P (SP), a possible mediator of stress-induced inflammatory reactions, and interleukin-1 receptor antagonist (IL-1ra). Twelve healthy male subjects (mean age 27 yrs.) were subjected to the psychosocial stress test "Trier Social Stress Test" (TSST) and a resting control condition. Blood and saliva samples were taken before, as well as 1, 20, 45, and 90 min after TSST or rest, respectively. Salivary cortisol and plasma SP and IL-1ra were measured using immunoassays, salivary alpha-amylase (sAA) was measured by an enzyme kinetic method, and plasma epinephrine (E) and norepinephrine (NE) were measured by HPLC. The TSST induced immediate increases of E, NE, and sAA, and a delayed increase of free cortisol. Plasma IL-1ra showed an even further delayed peak at 90 min after stress. Plasma levels of SP did not respond to stress. No significant associations between changes of stress hormones and IL-1ra or SP were found. We conclude that substance P, epinephrine, and norepinephrine are probably not involved in mediating peripheral inflammation following psychosocial stress, at least with respect to IL-1ra. Further studies have to reveal the mechanisms involved in the stress-induced up regulation of IL-1ra.     

The complete primary structure of the human snRNP E protein.

The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.     

DNA sequence of a human Sm autoimmune antigen. The multigene family contains a processed pseudogene

The sequence of a complementary DNA clone coding for a human autoimmune antigen has been determined. This DNA sequence predicts the amino acid sequence of a small protein ("E") which is associated with small nuclear RNA in human cells. Analysis of the predicted protein sequence suggests that the E protein is not closely related to other nucleic acid binding proteins. Screening of a human genomic DNA library has led to the isolation of several members of the E protein multigene family. Sequence analysis of one member of this family reveals that it is flanked by direct repeats and contains several mutations. One of these mutations, an insertion, terminates the long open reading frame. These features are compatible with the designation of this sequence as a processed pseudogene.     

Solid-phase processing of U2 snRNA precursors

HeLa cell cytoplasmic extracts contain both precursors to small nuclear RNA (snRNA) U2 and an activity that is capable of trimming these snRNA precursors to the size of mature U2. The substrate for this RNA processing reaction is the ribonucleoprotein complex containing pre-U2 RNA. To circumvent the difficulty of biochemically isolating pre-U2 ribonucleoprotein (pre-U2 RNP) complexes for use as substrate for the analysis of the processing activity, we have developed a procedure for the processing of pre-U2 RNP complexes that have been immobilized on anti-Sm antibody/protein A-Sepharose columns. When the immobilized [3H]uridine-labeled substrate RNP complexes are incubated at 37 degrees C with unlabeled cytoplasmic extracts from HeLa cells, labeled molecules the size of mature U2 are produced in a linear fashion for up to 3 h. Similar results are obtained when substrate pre-U2 RNPs are immobilized with an anti-2,2,7-trimethylguanosine antibody. Thus, accurate processing of the 3' termini of U2 precursors occurs on the antibody columns. Incubation with buffer alone does not result in the production of mature-sized U2, indicating that the processing activity is not intrinsic to the pre-U2 RNP. Using this assay procedure, we have demonstrated that the processing activity is destroyed by trypsin or by preincubation at 65 degrees C but is resistant to treatment with micrococcal nuclease. These results are compatible with the conclusion that the processing activity is a classical enzyme that does not contain a nuclease-sensitive essential RNA component.     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.