Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.     

Management of Spontaneously Ruptured Hepatocellular Carcinomas in the Radiofrequency Ablation Era

Background and aim

Spontaneous rupture of hepatocellular carcinoma (HCC) carries a high mortality. The use of radiofrequency ablation (RFA) in recent years has enriched the armamentarium for hemostasis of spontaneously ruptured HCCs but its results have not been documented. This study investigated the prognosis and outcome of spontaneous rupture of HCC as well as the results of using RFA for hemostasis.

Patients and method

From January 1991 to December 2010, 5283 patients were diagnosed with HCC at our hospital, and 189 of them had spontaneous rupture of HCCs. They were grouped under two periods: period 1, 1991–2000, n = 70; period 2, 2001–2010, n = 119. RFA was available in period 2 only.

Results

Hepatitis B virus infection was predominant in both periods. Surgical hemostasis was mainly achieved by hepatic artery ligation in period 1 and by RFA in period 2. The 30-day hospital mortality after surgical treatment was 55.6% (n = 18) in period 1 and 19.2% (n = 26) in period 2 (p = 0.012). Multivariate analysis identified 4 independent factors for better overall survival, namely, hemostasis by transarterial chemoembolization (hazard ratio 0.516, 95% confidence interval 0.354–0.751), hemostasis by RFA (hazard ratio 0.431, 95% confidence interval 0.236–0.790), having surgery as a subsequent treatment (hazard ratio 0.305, 95% confidence interval 0.186–0.498), and a serum total bilirubin level <19 umol/L (hazard ratio 1.596, 95% confidence interval 1.137–2.241).

Conclusion

The use of RFA for hemostasis during laparotomy greatly reduced the hospital mortality rate when compared with conventional hepatic artery ligation.     

Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides.

Three truncated fragments, harboring different sushi domains, namely, sushi123, sushi1, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushi1 and 3 each has a high-affinity LPS binding site with K:(d) of 10(-9) to 10(-10) M. Positive cooperativity in sushi123 resulted in a 1000-fold increase in K:(d)2. The core LPS binding region of sushi1 and 3 reside in two 34-mer peptides, S1 and S3. A rigidly held disulfide-bonded structure is not essential but is important for LPS binding, as confirmed by a 100- to 10000-fold decrease in affinity. Both S1 and S3 can inhibit LAL reaction and LPS-induced hTNF-alpha secretion with different potency. LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hill's coefficient of 2.42. The LPS binding by S3 is independent and noncooperative. The modified SDelta1 and SDelta3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10-fold improvement. Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS. Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an alpha-helical or beta-sheet structure. Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding. The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.     

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.     

The purification and properties of the scaffolding protein of bacteriophage lambda.

The Nu3 gene of bacteriophage lambda resides within a cluster of genes that specify structural components of the bacteriophage head. Previous experiments indicate that the Nu3 gene product (gpNu3) is associated with immature proheads but is not detectable in mature proheads or bacteriophage particles, hence its classification as a scaffolding protein. The Nu3 gene has been cloned and overexpressed, and its protein product has been purified. The purified protein is biologically active, as demonstrated by its ability to complement a gpNu3-deficient extract in an in vitro assembly reaction. The sequence of the amino terminus of the protein indicates that translation of Nu3 starts at nucleotide position 5,342 on the standard lambda DNA sequence, yielding a protein with a calculated Mr of 13,396. A combination of gel exclusion chromatography and velocity sedimentation gradient data indicates that gpNu3 possesses an unusually elongated shape.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.