Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 degrees C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.     

ENVIRONMENTAL MODULATION OF KARLOTOXIN LEVELS IN STRAINS OF THE COSMOPOLITAN DINOFLAGELLATE,KARLODINIUM VENEFICUM (DINOPHYCEAE)1

We examined the influence of N or P depletion, alternate N‐ or P‐sources, salinity, and temperature on karlotoxin (KmTx) production in strains of Karlodinium veneficum (D. Ballant.) J. Larsen, an ichthyotoxic dinoflagellate that shows a high degree of variability of toxicity in situ. The six strains examined represented KmTx 1 (CCMP 1974, MD 2) and KmTx 2 (CCMP 2064, CCMP 2283, MBM1) producers, and one strain that did not produce detectable karlotoxin under nutrient‐replete growth conditions (MD 5). We hypothesized that growth‐limiting conditions would result in higher cell quotas of karlotoxin. KmTx was present in toxic strains during all growth phases and increased in stationary and senescent phase cultures under low N or P, generally 2‐ to 5‐fold but with some observations in the 10‐ to 15‐fold range. No karlotoxin was observed under low‐N or low‐P conditions in the nontoxic strain MD 5. Nutrient‐quality (NO₃, NH₄, urea, and glycerophosphate) did not affect growth rate, but growth on NH₄ produced 2‐ to 3‐fold higher cellular toxicity and a 50% higher ratio of KmTx 1‐1:KmTx 1‐3 in CCMP 1974. CCMP 1974 showed higher cellular toxicity at low salinity (≤5 ppt) and high temperature (25°C). Our results suggested that given the presence of a toxic strain of K. veneficum in situ, the existence of environmental conditions that favor cellular accumulation of karlotoxin is likely a significant factor underlying K. veneficum–related fish kills that require both high cell densities (10⁴ · mL^?1) and high cellular toxin quotas relative to those generally observed in nutrient‐replete cultures.     

Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.     

COPI budding within the Golgi stack

The Golgi serves as a hub for intracellular membrane traffic in the eukaryotic cell. Transport within the early secretory pathway, that is within the Golgi and from the Golgi to the endoplasmic reticulum, is mediated by COPI-coated vesicles. The COPI coat shares structural features with the clathrin coat, but differs in the mechanisms of cargo sorting and vesicle formation. The small GTPase Arf1 initiates coating on activation and recruits en bloc the stable heptameric protein complex coatomer that resembles the inner and the outer shells of clathrin-coated vesicles. Different binding sites exist in coatomer for membrane machinery and for the sorting of various classes of cargo proteins. During the budding of a COPI vesicle, lipids are sorted to give a liquid-disordered phase composition. For the release of a COPI-coated vesicle, coatomer and Arf cooperate to mediate membrane separation.     

Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats

The effect of antiestrogens (MER-25, ICI-46474, and cis-clomiphene) and aromatase inhibitors (5-α-androstanedione, metopirone, and aminoglutethimide) on androgen induced copulatory behavior was tested in sexually inexperienced castrated male tats. Daily injections of 1 mg testosterone (T) for 21 days induced sexual activity in most subjects (61% mounting). Daily pretreatment with MER-25 or cis-clomiphene at three dose levels did not block the behavioral response to T. ICI-46474 at the high dose level (1 mg/kg) elicited a significant depressory effect on the sexual behavior of the T treated castrated rats. A single injection of 6 mg testosterone propionate (TP) induced mounting behavior in 56% of the tested rats within 120 hr. Treatment with metopirone or 5 α-androstanedione (injections every 12 hr for 96 hr) did not inhibit the response to TP. By contrast, aminoglutethimide (5 or 15 mg every 12 hr for 96 hr) abolished the behavioral response to androgen.     

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

An ultrasensitive colorimetric assay for manganese

An ultrasensitive colorimetric assay for manganese is described. It is based upon the catalysis, by Mn(II), of the photochemical oxidation of o-dianisidine, sensitized by riboflavin. Catalase increases the Mn(II)-catalyzed rate of photosensitized oxidation of dianisidine to the bisazobiphenyl, while superoxide dismutase inhibits the rate. The mechanism appears to involve oxidation of Mn(II) by O2-, followed by oxidation of dianisidine by MnO2+ in equilibrium Mn(III). Cu(II) interferes, but Zn(II), Fe(II), Fe(III), Co(II), and Ni(II) do not. Chelating agents and thiol reductants also interfere. Interference by Cu(II) can be overcome by the addition of cyanide, while interference by organic compounds can be surmounted by wet ashing. This assay provides a linear response to Mn(II) over the range 10-2500 nM. The limit of detection was 5 nM Mn(II).     

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site

Extremophiles - Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly...