Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection

A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.     

Molecular definition of a chromosome 9p21 germ-line deletion in a woman with multiple melanomas and a plexiform neurofibroma: implications for 9p tumor-suppressor gene(s).

Cutaneous malignant melanoma (CMM) is often familial, but the mode of inheritance and the chromosomal location of melanoma susceptibility locus are controversial. Identification of a 34-year-old woman with eight primary malignant melanomas, multiple atypical moles, and a de novo constitutional cytogenetic rearrangement involving chromosomes 5p and 9p suggested the presence of a melanoma predisposition gene at one of these locations. A high-resolution karyotype showed a partial deletion of a dark-staining Giemsa band, either 5p14 or 9p21. The patient was heterozygous for five 5p14 RFLPs. In situ hybridization with D9S3 indicated that this 9p21 marker was deleted. Gene dosage studies demonstrated the deletion of two more distal 9p21 markers, D9S126 and IFNA. In addition, she was hemizygous for the more proximal 9p21 short tandem-repeat polymorphism at D9S104. D9S18, D9S19, and D9S33 were retained, localizing the deletion to 9p21 between D9S19 on the proximal side and D9S33 on the distal side. Pulsed-field gel electrophoresis with D9S19 and D9S33 did not reveal any junction fragments in the patient's DNA. This germ-line deletion suggests that mutations in a 9p21 gene may initiate melanoma tumorigenesis.     

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.     

Block of outward current in cardiac purkinje fibers by injection of quaternary ammonium ions

We have studied the effects of iontophoretic injection of the quaternary ammonium compounds tetraethylammonium (TEA) and tetrabutylammonium (TBA) in cardiac purkinje fibers. We find that TBA(+) is a more effective blocker than TEA(+), but injection of either compound reduces the time-dependent outward plateau currents, transient outward current (I(to)), and the delayed rectifier (I(x)). Our findings provide evidence that these outward cardiac currents are carried by channels that in some respects are pharmacologically similar to squid axon potassium channels. We demonstrate that this procedure is a new tool that can be useful in the analysis of membrane currents in the heart.     

First record of lily thrips, Liothrips vaneeckei Priesner, in Australia (Thysanoptera: Phlaeothripidae)

The first Australian record of the lily thrips, Liothrips vaneeckei Priesner, is reported from a bulb farm in Warragul South, Victoria. It is an occasional pest of Lilium bulbs, both in the field and in storage, particularly in the USA and several European countries, and is also infrequently found in considerable numbers on the corms of orchids.     

Inactivation of MS-2 phage and poliovirus in groundwater

Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27 degrees C, complete virus inactivation took place in eight days. The temperature was then shifted to 4 degrees C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle.