A new class of antihypertensive neutral lipid: 1-alkyl-2-acetyl-sn-glycerols, a precursor of platelet activating factor

A new type of neutral lipid is described that possesses hypotensive activity in genetic hypertensive (SHR) and normotensive (WKY) rats. 1-Alkyl-2-acetyl-sn-glycerols and 1-alkyl-2-propionyl-sn-glycerols are both equally effective in eliciting the hypotensive response. Requirement for the 1-alkyl and 2-acetyl or 2-propionyl structure of the active isomer was documented by the negative responses obtained with closely related neutral lipid analogs (1-alkyl-2-acyl-, 1-alkyl-3-acetyl-, 1-acyl-2-acetyl-, 1-alkyl-2,3-diacetyl-, and 1-alkyl-glycerols). Although less potent than PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), the 1-alkyl-2-acetyl-sn-glycerols produce a response of significantly longer duration and may have fewer immediate side effects than PAF. The mechanism for the biological activity is unknown; however, we have demonstrated previously that the enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycerols to PAF occurs via a specific cholinephosphotransferase and therefore the observed blood pressure response might be due to the conversion of the neutral lipid precursor to PAF in vivo.     

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [³H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.     

Differential regulation of a novel variant of the alpha(6) integrin, alpha(6p).

We have reported previously the existence of an M(r) 70,000 form of the alpha(6) integrin called alpha(6p) in a variety of human epithelial cell lines. Four different experimental conditions were used to examine the regulation of alpha(6) and alpha(6p) integrin. The production of the alpha(6) integrin was decreased by 45% using a protein translation inhibitor (2.25 microM puromycin), whereas production of the alpha(6p) variant was unaffected. The alpha(6p) variant was decreased 60% by actin depolymerization (10 microM cytochalasin D) corresponding to a decrease in its surface expression, whereas alpha(6) integrin production was unaffected. The alpha(6p) variant was resistant to endoglycosidase H treatment, whereas the alpha(6) integrin was both sensitive and resistant to endoglycosidase H treatment, indicating retention in the endoplasmic reticulum and processing through the Golgi apparatus. Additionally, digestion by endoglycosidase F demonstrated both alpha(6p) and alpha(6) integrin contained NH(2)-linked glycosylations and both shifted M(r) approximately 10,000 on enzymatic digestion. Finally, inhibition of serine/threonine phosphatases by either calyculin A (15 nM) or okadaic acid (62 microM) did not affect alpha(6p), whereas the production of alpha(6) integrin was decreased by 50%. These data suggest that the production of the alpha(6p) variant is distinct from alpha(6) integrin and may involve a post-translational processing event at the cell surface.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

The effect of heat and radiation on the initiation and elongation processes of DNA synthesis

The pH step alkaline elution and alkaline sucrose gradient techniques were utilized to evaluate alterations in DNA replication (initiation and elongation) induced by heat and low dose X-irradiation is synchronized Chinese hamster ovary cells. The initiation and elongation process of DNA synthesis were radioresistant at the G1/S boundary (4 hours after mitosis) while in mid S phase (9 hours after mitosis) DNA initiation and elongation were sensitive to X-irradiation. The initiation and elongation processes of DNA synthesis which were radiation resistant at the G1/S boundary could be inhibited by a hyperthermia treatment (43 degrees C for 1 hour beginning at 4 hours after mitosis). The impairment of initiation in the heated cells was maintained through late S phase while that of elongation was reversible as judged by full recovery at 15 hours after mitosis. These data suggest that the known synergistic lethality of heat and radiation may be mediated by an impairment of initiation of DNA synthesis.     

A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.     

Improved ecdysone receptor-based inducible gene regulation system.

To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus musculus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn-off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively.