Construction of first-generation lactococcal integrative cloning vectors

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (ery ^r) gene obtained from L. lactis IL1837. Integration of the ery ^r gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nis ^r) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nis ^r fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for >200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (Nis ^r ) transformant suggested that the integrated nis ^r gene may be amplifying within the host chromosome. Correspondence to: S. K. Harlander     

Morphological variation in populations of the diatom Asterionella ralfsii W. Smith from Nova Scotia,Canada

Preliminary investigations of the diatom genus Asterionella ralfsii W. Smith from Kejimkujik National Park, Nova Scotia indicate that its morphology differs from other reported forms. Mean cell length increased between the spring and the fall. Bimodal distribution of length classes occurred in several lakes and was not related to measured environmental variables. The need for further work on this species in this and other geographical areas is considered.     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Consistent Errors in First Strand cDNA Due to Random Hexamer Mispriming

Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.     

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

Liver cells secrete the plasma form of platelet-activating factor acetylhydrolase.

Platelet-activating factor (PAF) is a phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) with diverse physiological effects. It has been implicated as a mediator of inflammation, allergy, shock, and thrombosis. Plasma contains an enzyme, PAF acetylhydrolase, that catalyzes the degradation of PAF, and the level of this enzyme may regulate the concentration of PAF in the blood and extracellular spaces under some conditions. Thus, the cellular source(s) of this enzyme and the factors that regulate its synthesis and secretion are issues that may have important physiological and pathological implications. We found that cultures of Hep G2, a human hepatocarcinoma line, secreted PAF acetylhydrolase activity. Optimal secretion occurred in medium that contained serum, and the newly secreted PAF acetylhydrolase was associated with high density and low density lipoproteins (LDL and HDL, respectively), just as the enzyme is in plasma. In the absence of serum. PAF acetylhydrolase was secreted with a particle that had a density similar to HDL. Apolipoproteins B and E were found in the same fractions. We tested the effects of a variety of hormones on the secretion of PAF acetylhydrolase and found that secretion was inhibited by 17 alpha-ethynylestradiol with a maximal effect at 30 microM. This may account for the observation of others that estrogens reduce the activity of PAF acetylhydrolase in the plasma. The PAF acetylhydrolase secreted by Hep G2 cells appeared to be identical to the enzyme in human plasma based on substrate specificity, association with LDL and HDL, response to inhibitors, and reactivity with antibodies against the plasma PAF acetylhydrolase. In conclusion, we have demonstrated that hepatocytes in culture secrete a PAF acetylhydrolase that is apparently identical to the plasma form. The secretion is constitutive but may also be regulated in response to hormonal stimulation.     

Quantification of roots and seeds in soil with real-time PCR

Study of roots and associated organisms in soil particularly in mixed plant populations, such as pastures, is limited by difficulties in quantification of root growth and function. The research evaluated the potential of DNA quantification by real-time PCR to improve our capacity to study and understand roots in such contexts. Probes and primers were developed for two common pasture species, Trifolium subterraneum and Lolium perenne (and closely related Lolium spp.), and evaluated for specificity and sensitivity in TaqMan assays on DNA extracted from soil. Further experiments examined the ability to detect DNA in dead roots, the changes in root DNA levels of plants defoliated or treated with herbicide and the relationship between DNA and root dry weight for single and mixed plant species grown in pots. T. subterraneum DNA/PCR 200 fg/µl was detected at 17.5 cycles and L. perenne at 19.5 cycles. The assay for T. subterraneum was species specific but the L. perenne assay, as anticipated from the choice of probe, also detected some closely related species. The assays were sensitive and capable of detecting equivalent to <2 mg roots/kg of dry soil and able to quantify targets in mixed populations. DNA concentration varied with plant age and genotype and DNA in dead roots found to decay rapidly over a few days. DNA concentrations in roots were found to respond more rapidly to defoliation and herbicide treatments than root mass. This approach appears to offer a new way to study roots in soil and indicates that quantifying root DNA could provide insights into root function and responses not readily provided by other methods.