Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation — FH469→Stop

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469_Trp(TGG). The GA mutation changes this codon into the amber stop codon, and is referred to as FH_469Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH_469Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.     

Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.     

Biogeneration of 2-phenylethanol and 2-phenylethylacetate important aroma components

Summary 2-Phenylethanol and 2-phenylethylacetate important aroma components are obtained in substantial amounts whenHansenula anomala CBS 110 andKloeckera saturnus CBS 5761 are grown with L-phenylalanine as sole nitrogen source. Up to 2 g/L of mixtures of alcohol and ester are obtained. While in the first microorganism the alcohol predominates, in the second the acetate is formed almost exclusively. Experiments with 1-¹³C-d-Glucose show complete incorporation of the label into the methyl group of the acetate.     

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.     

Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.     

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.