Search

There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.

A rapid, specific, stability-indicating high-performance liquid chromatographic (HPLC) method has been developed for the assay of Ampicillin in Ampicillin Trihydrate bulk, capsules and oral suspensions and Sodium Ampicillin bulk and injectables. The assay is specific for Ampicillin in the presence of possible contaminants; Penicillin V, Phenylglycine, and 6-Aminopenicillanic Acid (6-APA); the degradation product, Penicilloic Acid of Ampicillin; and all excipients present in the formulations assayed. Ampicillin, Ampicillin formulations, and formulation excipient blends were force-degraded to further demonstrate specificity. The assay is precise, accurate, linear over the range 50% to 125% of expected Ampicillin sample level, and stability-indicating toward the described thermal, acid, base, aqueous, and light degradations. The procedure employs an ion-pairing eluent with UV detection at 254 nm. Ampicillin Trihydrate and Sodium Ampicillin bulk are stable in assay diluent for six hours allowing the ...

A high-pressure liquid chromatographic method for the assay of L-Lysine is described. L-lysine was derivatized by reacting with 2,4-dinitrofluorobenzene. A fixed wavelength detector (254 nm) and a LiChrosorb RP-18, 10 μm, Hibar-II column from MCB Manufacturing Chemists, Inc., was employed. The method is simple, precise, accurate and stability-indicating.

A rapid Paired-ion high-pressure liquid chromatographic method for the quantitative determination of guaifenesin, phenylpropanolamine hydrochloride, sodium benzoate and codeine phosphate in cough syrups was developed. Heptane sulfonic acid was used as a counter-ion in the mobile phase. A fixed wavelength detector (λ = 254 nm) and a μ-Bondapak phenyl column was employed. Total analysis time for all the active ingredients was 22 minutes.

A specific, stability-indicating, high-performance liquid chromatographic assay was developed for the determination of cephapirin, desacetyl cephapirin and cephapirin lactone in sodium cephapirin (cefadyl) bulk and injectables. The procedure uses a muBondapak C18 column and a mobile phase of dimethylformamide-acetic acid-potassium hydroxide in water. UV detection at 254 nm is used for quantitation with acetanilide used as the internal standard. The assay is precise, accurate and linear over the range of 100-300 micrograms/ml for cephapirin and over the range of 2-6 micrograms/ml for desacetyl cephapirin and cephapirin lactone. The assay is also stability-indicating for the described thermal, acid, base, aqueous and accelerated light degradations.

A rapid, ion-pair, high-pressure liquid chromatographic method for analysis of Dane Salt Potassium (-)-N-(1-Methoxycarbonylpropene-2-yl)-p-Hydroxy-phenylglycine was developed. Tetrabutylammonium hydroxide was used as a counter-ion in the mobile phase. A fixed wavelength detector (λ = 280 nm) and a μ-Bondapak C-18 column were employed. The percent relative range of the method (precision) was 0.6% (n = 3).

Simple and specific assays for etoposide, related impurities, and benzyl alcohol, as well as its degradation product, benzaldehyde, in injectable formulations were developed using high-performance liquid chromatography (HPLC). Etoposide, benzyl alcohol, benzaldehyde, and four etoposide impurities were determined with a mobile phase containing 26% acetonitrile in pH 4.0 acetate buffer with methyl p-aminobenzoate as the internal standard. Two etoposide precursors were additionally determined using 60% acetonitrile in the mobile phase with biphenyl as the internal standard. The assays were run sequentially. The etoposide assay had a total variability of less than 1% (RSD) and a recovery from placebo of 99.4%. Mean recovery of impurity standards spiked at the 2% level relative to the etoposide level was 98%. Recoveries for benzyl alcohol and benzaldehyde were 100.5% and 102.2%, respectively. Absorbance ratios generated from samples and compared with undegraded standards demonstrated specificity for etoposide. Similar response regression equations with correlation coefficients greater than 0.99 were obtained for etoposide and four impurities over the 2-8 micrograms/mL concentration range, indicating that an etoposide standard could be used to quantitate low impurity levels down to 0.2% relative to the etoposide injectable concentration. Benzaldehyde could be reliably detected at concentrations down to 0.5 micrograms/mL, equivalent to 0.16% of the benzyl alcohol level.

The analysis of tetracycline epimers in tetracycline preparations by high-performance liquid chromatography is described. The method uses a microparticulate phenyl column (3.9 mm i.d. X 30 cm) with a step gradient of 12-22% acetonitrile in 0.2 M phosphate buffer at pH 2.2. The analysis takes 22 min. The relative standard deviations of the method (2 sigma, n = 6) for the analysis of chlortetracycline, 4-epitetracycline, 4-epianhydrotetracycline, and anhydrotetracycline in tetracycline were +/- 3.68, +/- 4.47, +/- 7.60, and +/- 2.77 %, respectively.

Specific assays of tiodazosin and levulinic acid from tablet preparations were developed using high-performance liquid chromatography. Tiodazosin was determined with acetonitrile 50% (v/v) in pH 5.7 acetate buffer on a 300 X 3.9-mm i.d. microparticulate C-18 column using 254 nm detection with butylparaben as the internal standard. Levulinic acid was determined at 280 nm with 1 N acetic acid on a 300 X 3.9-mm i.d. microparticulate phenyl column using an external standard. The assays were run sequentially. The relative standard deviations of sample variability (2 sigma, n = 6) and recoveries from synthetic preparation were +/- 1.6 and approximately 95%, respectively, for both assays.