Synthesis and growth regulator activity of fatty acyl derivatives of d-glucose and d-galactose

In an attempt to gain information about one or more components of the brassin complex, fatty acid esters of d-glucose and d-galactose were prepared and tested for growth regulator activity in a bean hypocotyl bioassay. 4-O-Acyl-d-glucoses and, perhaps, 1-O-acyl- d-galactoses had a similar qualitative activity to that of the brassin complex. 3-O-Acyl- d-galactoses inhibited elongation of bean hypocotyls and stimulated rooting. 3- And 6-O- acyl-d-glucoses both stimulated and inhibited elongation, depending on the source of fatty acids; in both cases, stimulation was observed when safflower oil was used as the source of fatty acids and inhibition was observed when peanut oil was used as the source of fatty acids. Fatty alkyl β-d-galactopyranosides were inactive.     

ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Assessment of thin-layer breast aspirates for immunocytochemical evaluation of HER2 status

OBJECTIVE: To determine whether immunocytochemistry (ICC) for HER2 on ThinPrep (TP)-processed breast fine needle aspiration biopsies (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) is comparable to the findings of immunohistochemistry on corresponding surgically removed tissue. STUDY DESIGN: Immunostaining was performed on 63 malignant breast fine needle aspirates and compared to immunostaining on paraffin sections (PSs) from the subsequent biopsies. The HercepTest (Dako, Carpinteria, California, U.S.A.) and TAB250 antibodies were utilized. Cases in which the TP and paraffin HER2 results did not correlate were further assessed for gene amplification by differential polymerase chain reaction (dPCR). RESULTS: HER2 overexpression was found in 9 of the 63 cases (14%). TAB250 had higher specificity on PS versus TP (P = .008), and TAB250 had higher specificity on PS versus the HercepTest on PS and TP (P = .004 and .0001, respectively). CONCLUSION: HER2 immunostaining with both the HercepTest and TAB250 on TP is unreliable due to low specificity (72% and 83% for HercepTest and TAB250, respectively). However, both antibodies have high sensitivity (89% and 100%, respectively); suggesting that this method may have some utility as a preliminary screening test for HER2 status. Negative HER2 staining by ICC is highly predictive of the absence of HER2 overexpression, whereas positive HER2 staining on TP would require further validation by either dPCR of fluorescence in situ hybridization.     

Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.     

Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions

Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date. One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal. In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures. Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity. This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media. These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions. To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80. At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement. Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions. After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale.     

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.     

Genomic growth hormone gene polymorphisms in native Chinese chickens

Chicken growth hormone (cGH), a polypeptide hormone synthesized in and secreted by the pituitary gland, is involved in a wide variety of physiological functions such as growth, body composition, egg production, aging, and reproduction. Chicken growth hormone polymorphisms have been reported to be associated with certain phenotypes. Our objective is to investigate the GH gene polymorphism in selected strains of native Chinese chickens. Yellow Wai Chow GH gene was characterized by sequencing and was found to have one silent substitution, 31 insertions, and other substitutions spread among the introns. In addition, a novel Mspl site has been identified and characterized in the first intron. Allele frequencies of the intron 1 polymorphism were characterized among 28 populations of native Chinese chickens. Thus, polymorphism of the cGH gene may be useful in phylogenetic analysis, as well as in the design of breeding programs.