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Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

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A rigorous measurement of the photoluminescence quantum yield (PLQY) of three luminescent solid state organic material systems is presented. Poly(9,9-dioctylfluorene), perylene (2.97 M in poly(methyl methacrylate)), and perylene red (0.78 M in poly(methyl methacrylate)), were measured using a Ti:sapphire laser yielding 47 ± 3%, 79 ± 3%, and 51 ± 2%, respectively. A GaN diode laser with differing variability was used to measure the PLQY for perylene and perylene red yielding 71 ± 1% and 53 ± 2%, respectively. Variations due to sample preparation (<0.5%), sample degradation (none), and measurement system repeatability (Ti:sapphire ≈2%, GaN ≈1%) have been determined for each material. Variance in laser intensity is found to be the largest source of error which upon propagation to the PLQY, agrees closely with the uncertainty found by means of the rigorous statistics. This suggests reduction of laser intensity variation could allow much greater precision in absolute determinations of PLQY. Some small systematic bias from calibration and self-absorption corrections cannot be ruled out. The current limit of precision for this measurement is ±1% using the more stable GaN laser though this apparently depends on the material and sample fabrication.

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Current technologies for the purification of supercoiled plasmids are limited. The use of cesium chloride gradients in the presence of ethidium bromide is time consuming, labor intensive, requires the use of known mutagens and is not conducive to large scale. As a result, first-generation high-performance liquid chromatography (HPLC) methods based on anion-exchange and size exclusion have been developed but are difficult to accommodate production at large scale and still result in compromised purity (1,2). The success of DNA vaccines in animal models and the initiation of human trials (3,4) has led to a need to increase the level of supercoiled plasmid purity as well as the methodology utilized to produce these plasmids at large scale. Several parameters of the purification process need to be addressed: • The ability to prepare supercoiled plasmid at purity levels acceptable for clinical material. • The ability to prepare clinical grade supercoiled plasmid that will be scalable in order to produce gram quantities of product. • The ability to prepare clinical grade supercoiled plasmid in accordance with cGMP principles. • The ability to develop validated assays to assess purity, yield, and contamination levels.

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Applications for a new polymer resin, PolyFlo, are described for both the small-scale and large-scale purification of synthetic oligodeoxyribonucleotides varying in length from 18-41 bases. The unique properties of this innovative resin provide > 95% purified full-length oligodeoxyribonucleotides with greater than 90% yield starting from either crude trityl-on or trityl-off unmodified as well as base (biotin)- or backbone (e.g., phosphorothioate)-modified products. Full biological activity of recovered nucleic acid is retained, and the resin is capable of removing contaminating endotoxins during purification. The resin performance is predictable and reliable. The resin can be regenerated easily and is particularly economic when employed directly in ammonia or with the trityl-off option. PolyFlo meets the requirements of current Good Manufacturing Practices.

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Adenosine is a purine nucleoside that impairs conduction through the AV node and is thus effective in terminating tachycardias involving the AV node. Gaining acceptance as the drug of choice for neonatal supraventricular tachycardia (SVT), it is given IV as a rapid bolus with an initial dose of 0.05 mg/kg and can be increased in increments of 0.05 mg/kg every one to two minutes until termination of SVT (to a maximum of 0.25 mg/kg). Because of its half-life of 0.6 to 10 seconds, adenosine will not prevent reinitiation of SVT, therefore other medications should be considered if prophylaxis is required. An advantage of the short half-life is the transient nature of adverse effects, which can include flushing, nondistressing alterations in respiratory pattern, irritability, sinus bradycardia, and varying degrees of AV block. Administration to critically ill infants, including those requiring mechanical ventilation, has been reported. The infant's blood pressure, electrocardiogram, respiratory status, and capillary refill should be monitored before, during, and after adenosine administration.

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Endonuclease V is the product of the denV gene of bacteriophage T4 and is responsible for the recognition and repair of pyrimidine dimers due to UV irradiation of DNA. This is accomplished by a two-step mechanism involving incision at the site of the lesion followed by cleavage of the phosphate backbone. In order to better understand this molecule, and to validate our new mutagenesis procedure, we have constructed a series of random mutations within the region Ala116-->Lys121 using a random targeted mutagenesis procedure developed for this study. The results presented here suggest an important role for this region in the stabilization of the thymine dimer-containing substrate. These mutants also confirm a direct correlation between survival and both DNA binding and pyrimidine dimer-DNA glycosylase activity. No such correlation exists between survival and AP lyase activity. The results are consistent with the recently published X-ray crystal structure.

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We have developed a set of cell lines to help distinguish the sequelae of specific lesions in DNA after UV irradiation. Irradiation results in two primary lesions: cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The contributions of each to mutation are considered utilizing a spectrum of cell lines with increasing abilities to repair these lesions. In particular, we focus on a revertant of the XP12Ro(M1) cell line from a patient with Xeroderma pigmentosum, XP129, which is capable of repairing (6-4) photoproducts but not cyclobutane dimers. We have successfully introduced the denV gene into these cells which confers the ability to repair cyclobutane dimers. By comparing the results of a shuttle vector mutation experiment with the vector pZ189, we can correlate specific mutations to specific lesions.

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We demonstrate the feasibility of using passive host-cell reactivation of a shuttle-vector pRSVcat to detect cloned DNA-repair genes. As models, a transient expression vector, pRSVdenV, and a positive-selection vector, pRSVdenV/SVgpt, were constructed containing the T4 coliphage denV gene, coding for an ultraviolet-specific endonuclease, under promotion of the Rous sarcoma virus (RSV) long-terminal repeat. Cotransfection of one or three copies of pRSVdenV per UV-irradiated pRSVcat molecule into xeroderma pigmentosum (XP) cells (XP12Ro[M1]) resulted in a dramatic increase in transient expression of chloramphenicol acetyl transferase (CAT) activity. XP clones stable transformed by pRSVdenV/SVgpt but not the parent cell line rescued CAT activity from this UV-irradiated reporter gene. The ability to express CAT activity from a UV-irradiated pRSVcat correlated with the presence of the structural denV gene as detected by Southern blot analysis. Post-UV irradiation colony-forming ability and DNA nucleotide excision-repair synthesis were partially restored in XP clones which rescued CAT activity. These results demonstrate the feasibility of using the cloned denV gene with its well characterized pyrimidine cyclobutane dimer-specific endonuclease activity to reconstitute UV-induced DNA repair in human cells deficient in DNA repair. Measuring CAT expression from pRSVcat affords a rapid, sensitive procedure to screen for functional cloned DNA-repair genes and to test mutant cells for defects in DNA repair.

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In this paper we report both transient and stable complementation of pyrimidine dimer repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4, coding for endonuclease V, a dimer-specific DNA glycosylase. Cotransfection with pRSVdenV in SV40-transformed XP12RO(M1) cells (complementation group A) restored transient expression of an indicator plasmid (pRSVcat) bearing a UV-inactivated chloramphenicol acetyltransferase (cat) gene. In addition, XP12RO(M1) clones stably transformed by pRSVdenV-SVgpt expressed transient chloramphenicol acetyltransferase activity when transfected with UV-inactivated pRSVcat plasmid. These clones also showed partial restoration of colony forming ability and excision repair synthesis after UV irradiation. Immunofluorescence, using an endonuclease V polyclonal antibody, showed the presence of the phage glycosylase in stably transformed xeroderma pigmentosum cells. The cotransfection assay affords a rapid, sensitive procedure to screen for functional cloned DNA repair genes and to test mutant cells for the deficiency of specific steps in DNA repair, such as incision.

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The repeat dose toxicity of various liposomal formulations containing amphotericin B has been determined in mice. In general, small liposomes (e.g. 100-150 nm) were found to be more toxic than their large counterparts (e.g. about 2000 nm). However, the repeat dose toxicity of small liposomes could be diminished substantially by the inclusion of sterol (i.e. ergosterol) into the liposomal membranes. Tissue accumulation studies of amphotericin B after repeat dosing may be a useful adjunct to formulation development.

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BRL 20459 is a novel compound which displays anti-inflammatory activity when applied topically in the croton oil and cantharadin rat ear inflammation models. The compound does not inhibit uv-induced erythema in the guinea-pig or granuloma formation in the cotton pellet test in the rat. BRL 20459 does not inhibit prostaglandin synthesis nor does it interact with corticosteroid receptors in the thymus. In contrast to hydrocortisone, BRL 20459 did not cause thymus involution or reduce body weight gain in rats. BRL 20459 would seem to have a different mechanism of action to hydrocortisone, but this mechanism is as yet unknown.

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Nitrocellulose discs were implanted subcutaneously into mice and cell accumulation measured 96 hours later. Cell accumulation was inhibited by the anti-inflammatory steroids hydrocortisone, dexamethasone and triamcinolone acetonide, but higher doses of the latter two compounds were required than are normally used in rat models of inflammation. Cell accumulation was not inhibited by the cyclo-oxygenase inhibitors naproxen, indomethacin, tolmetin or aspirin, nor by the dual cyclo-oxygenase and lipoxygenase inhibitors BW 755C and benoxaprofen. Anti-rheumatic drugs such as D-penicillamine, dapsone and colchicine and the metal chelator 1,10 phenanthroline were all inactive on cell accumulation in this model.

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An injection of thioglycollate into the peritoneal cavity of mice produced a peak of exudate at 4 h, a peak of total leukocytes at 24 h when the predominant cell was the polymorphonuclear neutrophil, and a secondard macrophage phase beginning 2--3 days after thioglycollate. The release of N-acetylglucosaminidase (EC 3.2.1.30) into the peritoneal fluid paralleled the macrophage phase. The amount of enzyme released was related to the dose of thioglycollate. Neither zymosan (400 micrograms) nor endotoxin (30 micrograms) produced a marked inflammatory response when injected into the peritoneal cavity.

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Zymosan (400 microgram) and thioglycollate medium (0.1 ml of 2.4% w/v) have each been shown to induce a chronic inflammation when injected into the hamstring muscle of the mouse. The inflammation is characterized by an increase in muscle weight and N-acetylglycosaminidase concentrations. The response is not inhibited by oral treatment with hydrocortisone, dexamethasone or naproxen, but is inhibited by the local injection of methylprednisolone. The relevance of this model to polymyositis and fibrositis is discussed.

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