Control of Human PLP1 Expression Through Transcriptional Regulatory Elements and Alternatively Spliced Exons in Intron 1

Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin.     

Two mechanisms coordinate replication termination by the Escherichia coli Tus–Ter complex

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus–Ter complex. The T7 replisome was arrested at the non-permissive end of Tus–Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus–Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.     

Molecular polymorphisms in Palestinian Figs (Ficus carica L.) as revealed by Random Amplified Polymorphic DNA (RAPD)

The genetic diversity of 13 local Palestinian fig genotypes was investigated using RAPD markers. Among the 30 tested primers, 28 revealed various banding patterns and 2 generated no polymorphic bands. In addition, 13 primers (46.4%) produced good amplification products with high intensity and pattern stability. A total of 94 DNA fragments (loci), separated by electrophoresis on agarose gel were detected, ranging in size from 190 to 1300 bp. Of these fragments, 72 (76.6%) were polymorphic and 22 (23.4%) were monomorphic. A minimum of three and a maximum of eight DNA fragments were obtained using (OPH-02 and OPT-10) as well as (OPA-13, OPA-18 and OPY-07) primers respectively. The maximum percentage of polymorphic markers was 100.0 (Z-5, Z-12, and OPT-10) and the minimum was 60.0 (OPH-02). Primers OPY-07 and OPA-13 revealed high collective resolving power (Rp) values with 4.640 and 4.760 respectively and therefore, they were the most useful RAPD primers to assess the genetic diversity in the Palestinian figs. Genetic distance matrix showed an average distance range from 0.186 to 0.559 with a mean of 0.373. Thus, the cultivars tested in this study were characterized by large divergence at the DNA level. To our knowledge, this is the first report using RAPD marker to assess genetic diversity of Palestinian figs.     

Purification and Characterization of a Novel Anti-<Emphasis Type="Italic">Campylobacter</Emphasis> Bacteriocin Produced by <Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> DN317

The lactic acid bacteria (LAB) microbiota of Saudi chicken ceca was determined. From 60 samples, 204 isolates of lactic acid bacteria were obtained. Three isolates produced antimicrobial activities against Campylobacter jejuni, Listeria monocytogenes, and Bacillus subtilis. The isolate DN317, which had the highest activity against Campylobacter jejuni ATCC 33560, was identified as Lactobacillus curvatus (GenBank accession numbers: KX353849 and KX353850). Full inhibitory activity was observed after a 2-h incubation with the supernatant at pH values between 4 and 8. Only 16% of the activity was conserved after a treatment at 121 °C for 15 min. The use of proteinase K, pepsin, chymotrypsin, trypsin, papain, and lysozyme drastically reduced the antimicrobial activity. However, lipase, catalase, and lysozyme had no effect on this activity. The active peptide produced by Lactobacillus curvatus DN317 was purified by precipitation with an 80% saturated ammonium sulfate solution, and two steps of reversed phase HPLC on a C18 column. The molecular weight of this peptide was 4448 Da as determined by MALDI-ToF. N-terminal sequence analysis using Edman degradation revealed 47 amino acid residues (UniProt Knowledgebase accession number C0HK82) revealing homology with the amino acid sequences of sakacin P and curvaticin L442. The antimicrobial activity of the bacteriocin, namely curvaticin DN317, was found to be bacteriostatic against Campylobacter jejuni ATCC 33560. The use of microbial antagonism by LAB is one of the best ways to control microorganisms safely in foods. This result constitutes a reasonable advance in the antimicrobial field because of its potential applications in food technology.     

Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M(3) muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.     

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

Lipid composition and effect of amphotericin B on yeast cells of Paracoccidioides brasiliensis

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 g/mL.     

Cigarette smoking and face lift: conservative versus wide undermining

The effects of cigarette smoking on the skin flaps of the face lift procedure are discussed. Reported elsewhere is a significant incidence of skin slough in smokers with use of wide undermining techniques. This complication is thought to be due to the vasoconstrictive effects of nicotine on the peripheral circulation. Our group has employed a conservative bilateral undermining technique in 407 face lifts. Of these, 32.4 percent were smokers and 67.6 percent were nonsmokers. No cases of skin slough were encountered. Our conservative undermining technique is briefly discussed. Among its advantages are shorter operative time, use of less local and/or general anesthesia, less intraoperative bleeding, adequate exposure for SMAS and platysmal surgery, and snugger skin closure without the risk of flap necrosis. As shown by our statistics, it is a safer procedure in smokers than the usually performed more radical procedure.     

Methanol metabolism and ammonia assimilation in four methylophilns strains

Methanol assimilation and dissimilation pathways and ammonia assimilation pathway were investigated in four obligate methanol-utilizing bacteria through the detection of key enzymes. Both hexulose phosphate synthetase and hexulose phosphate isomerase, key enzymes of the ribulose monophosphate pathway (RMP) for methanol assimilation were detected whereas four key enzymes (hydroxy pyruvate reductase, isocitrate lyase, malyl-CoA-lyase and glyoxylate aminotransferase) that are characteristic of the serine assimilation pathway were absent. Key enzymes for the two methanol dissimilation pathways, the linear sequence enzymes formaldehyde and formate dehydrogenase and the RMP cyclic sequence enzymes glucose-6-phosphate dehydrogenase and 6-phosphate giuconate dehydrogenase were all detected. Ammonia was assimilated via the glutamate dehydrogenase pathway and not via the glutamine synthetase and glutamate synthase pathway.