Determination of argininosuccinate in normal blood serum and liver

Argininosuccinate has been determined in normal serum and liver extract of rats by chromatographic analysis on Dowex-1-acetate after a brief period of in vivo labeling with l-[U-¹⁴C] citrulline. The amounts found were within the range of other amino acid intermediates of the ornithine cycle, determined in the same samples. Both ¹⁴C and ninhydrin color were measured. Chromatography on Amberlite columns, subsequent to fractionation on Dowex, allowed determination of the other amino acids. Fractionation on Dowex-1-acetate provides a reliable and highly selective method for the analysis of minute amounts of argininosuccinate in biological samples containing other amino acids.     

Modified eudesmanolides and other sesquiterpene lactones from Wunderlichia mirabilis and Actinoseris polymorpha

Re-investigation of Wunderlichia mirabilis afforded three new modified eudesmanolides of the same type isolated previously from an Onoseris species, and minute amounts of a germacranolide for which a structure only could be proposed. Actinoseris polymorpha also contained a new germacranolide, the angetate of desacetyllaurenobiolide. Structures were elucidated by spectroscopic methods and by some chemical transformations. The significance of these findings is discussed briefly.     

Phosphoserine and phosphohydroxypyruvic Acid: evidence for their role as early intermediates in photosynthesis

Photosynthetic fixation of ¹⁴CO₂ in the bean Phaseolus vulgaris, cv. Pencil Pod Black Wax, resulted in the appearance of labeled compounds that were characterized as phosphoserine and phosphohydroxypyruvate by chromatographic separation and by the synthesis of chemical derivatives. In ¹⁴CO₂/¹²CO₂ pulse-chase experiments these metabolites demonstrated the rapid pool saturation and depletion of ¹⁴C characteristic of early intermediates in photosynthetic carbon fixation. They were present in sufficient amounts to account for about 35% of total carbon fixed in 1 minute.     

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans^9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP^C) and the infectious protein (PrP^Sc), an abnormally-folded isoform of PrP^{C 8}.One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation¹³. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP^Sc are poorly characterized due to their unusual conformation and aggregation states.PrP^Sc can seed the conversion of PrP^C to PrP^Scin vitro¹⁴. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP^Sc, at an accelerated rate, from a system containing excess amounts of PrP^C and minute amounts of the PrP^Sc seed¹⁹. This technique has proven to effectively recapitulate the species and strain specificity of PrP^Sc conversion from PrP^C, to emulate prion strain interference, and to amplify very low levels of PrP^Sc from infected tissues, fluids, and environmental samples^6,7,16,23 .This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.     

Microscale analysis of amino acids using gas chromatography–mass spectrometry after methyl chloroformate derivatization

To conduct studies of stable isotope incorporation and dilution in growing plants, a rapid microscale method for determination of amino acid profiles from minute amounts of plant samples was developed. The method involves solid-phase ion exchange followed by derivatization and analysis by gas chromatography–mass spectrometry (GC–MS). The procedure allowed the eluent to be derivatized directly with methyl chloroformate without sample lyophilization or other evaporation procedures. Sample extraction and derivatization required only ca. 30 min and quantification of the 19 amino acids eluted from the cation exchange solid-phase extraction step from a single cotyledon (0.4 mg fresh weight) or three etiolated 7-day-old Arabidopsis seedlings (0.1 mg fresh weight) was easily accomplished in the selected ion monitoring mode. This method was especially useful for monitoring mass isotopic distribution of amino acids as illustrated by Arabidopsis seedlings that had been labeled with deuterium oxide and ¹⁵N salts. Sample preparation was facile, rapid, economical, and the method is easily modified for integration into robotic systems for analysis with large numbers of samples.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.     

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

Formation of extracellular adenosine triphosphate by normal and neoplastic human cells in culture

Normal and neoplastic human cells in culture were suspended under isotonic conditions and incubated for one minute with the substrates, including ³²P-labelled inorganic phosphate, and cofactors of the glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions (complete medium), as well as with an incomplete medium lacking ADP, NAD⁺ and glyceraldehyde-3-phosphoric acid. The neoplastic cell types incubated in the complete medium synthesized three to six times more labelled ATP than the corresponding normal cells. In the incomplete medium only insignificant amounts of labelled ATP were formed during one-minute incubation by all types of cells. From other types of experiments it could be concluded that the labelled ATP, isolated from the cells incubated in the complete medium, was formed at the surface of the cell membranes. Only negligible amounts of enzymes engaged in the synthesis of ATP have leaked out from the cells.     

Ineupatorolide-like sesquiterpene lactones from Dittrichia viscosa

Dittrichia viscosa afforded, in addition to known compounds, minute amounts of nine sesquiterpene lactones, all closely related to ineupatorolide B     

Population Pharmacodynamic Modeling and Simulation of the Respiratory Effect of Acetazolamide in Decompensated COPD Patients

Background

Chronic obstructive pulmonary disease (COPD) patients may develop metabolic alkalosis during weaning from mechanical ventilation. Acetazolamide is one of the treatments used to reverse metabolic alkalosis.

Methods

619 time-respiratory (minute ventilation, tidal volume and respiratory rate) and 207 time-PaCO₂ observations were obtained from 68 invasively ventilated COPD patients. We modeled respiratory responses to acetazolamide in mechanically ventilated COPD patients and then simulated the effect of increased amounts of the drug.

Results

The effect of acetazolamide on minute ventilation and PaCO₂ levels was analyzed using a nonlinear mixed effect model. The effect of different ventilatory modes was assessed on the model. Only slightly increased minute ventilation without decreased PaCO₂ levels were observed in response to 250 to 500 mg of acetazolamide administered twice daily. Simulations indicated that higher acetazolamide dosage (>1000 mg daily) was required to significantly increase minute ventilation (P<.001 vs pre-acetazolamide administration). Based on our model, 1000 mg per day of acetazolamide would increase minute ventilation by >0.75 L min⁻¹ in 60% of the population. The model also predicts that 45% of patients would have a decrease of PaCO2>5 mmHg with doses of 1000 mg per day.

Conclusions

Simulations suggest that COPD patients might benefit from the respiratory stimulant effect after the administration of higher doses of acetazolamide.     

Determination of labeled adenine by means of adsorption on cellulose nitrate filters: a sensitive method for estimation of nucleosidase activity.

Cellulose nitrate filters strongly adsorb adenine up to 4.4 nmoles/cm² of the filter with a constant 90% efficiency. Based on this phenomenon a sensitive and fast method of estimation of labeled adenine in amounts over three orders of magnitude was developed. This method has been successfully applied to estimate the activity of minute amounts of a nucleosidase system, catalyzing the decomposition of ATP to adenine. Other purines and pyrimidines are also adsorbed by cellulose nitrate filters in the following order: Adenine > cytosine ? thymine > uracil > guanine. The affinity of corresponding nucleosides as well as nucleotides toward cellulose nitrate filters is greatly reduced. The kinetics of adsorption and desorption of [¹⁴C]adenine was studied with regard to temperature and time of adsorption, additives, source of the filters and their pore size. The mechanism of adsorption is discussed.     

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h⁻¹. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, α-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.     

The influence of dietary calcium and phosphorus on intestinal calcium transport in rats given vitamin D metabolites.

A new method for the simple analysis of methylated amino acids based on autoradiography is introduced. With this technique a survey of protein methylation in a prokaryote, Escherichia coli, and a eukaryote, fibroblasts in culture, was carried out in an attempt to identify, quantitate, and determine the subcellular localization of all the methylated amino acids found in the proteins of these organisms.In mammalian cells using an established mouse fibroblast line (3T3), we have found that nuclei-free and mitochondria-free cytoplasm contain readily detectable amounts of four identifiable methylated amino acids: N^?,N^?-dimethyllysine, N^?,N^?,N^?-trimethyllysine, N^G,N^G-dimethylarginine (or N^G-methylarginine), and N^G,N′^G-dimethylarginine. The crude nuclear pellet also contains these methylated amino acids, but in addition contains N^?-methyllysine and a new as yet unidentified methylated compound. Histones purified from these nuclei contain essentially the same array of methylated compounds.The ribosomal subunits of the mammalian cells contained only small amounts of the methylated amino acids; the 40S subunit contained a substantial amount of just one, N^G,N^G-dimethylarginine (or N^G-methylarginine), and smaller amounts of N^G,N′^G-dimethylarginine, and an as yet unidentified methylated compound. The 60S subunit contained even smaller amounts of methylated amino acids, 50% of which was N^?,N^?,N^?-trimethyllysine and smaller amounts of N^?-methyllysine, N^?,N^?-dimethyllysine, and N^G,N^G-dimethylarginine. These subunits also contained an as yet unidentified methylated compoundThese results were in marked contrast to those that we obtained with the prokaryote, Escherichia coli. Only the proteins of the 50S ribosomal subunit of the bacteria contained methylated amino acids. Of those present 50% was N^?,N^?,N^?-trimethyllysine, with the remainder distributed about equally between N^?-methyllysine and three unknowns, one of which is apparently the same as that found in the 60S subunit of the mouse fibroblasts. All of the N^?-methyllysine was apparently in the small acidic proteins, L7 and L12.     

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from escherichia coli

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca²⁺ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (>98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cell, and in investigating envelope-related synthesis and assembly processes.     

Comparison of bioassay and enzyme-linked immunosorbent assay for quantification of Spodoptera frugiperda nuclear polyhedrosis virus in soil

Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 × 10⁴ polyhedral inclusion bodies (PIB)/g sandy soil and <10 PIB/g soils with large amounts of silt or clay. The ELISA detected as few as 360 PIB/g in all three soil types, and absorbance values were inversely related to the amount of clay. Results of the bioassay and ELISA were significantly (P < 0.01) correlated for natural NPV from field samples of silt (R = 0.961) and sandy soil (R = 0.723). Soil samples from Louisiana pastures and corn fields contain up to 7.6 × 10⁴ PIB/g, and 2× 10⁴ PIB/g are commonly present.     

Characterization of three structurally-related 8–9 kDa monomeric peptides present in the Corpora cardiaca of Locusta: A revised structure for the neuroparsins

Recent developments in automated peptide microsequencing, liquid secondary-ion and electrospray mass spectrometry enable unambiguous primary structure determinations of minute amounts of biological material. We have used these methods in combination to characterize the predominant peptides from HPLC eluates of aqueous extracts of corpora cardiaca from adults of Locusta migratoria. Among the molecules or families of molecules clearly predominating in the extracts, we had previously characterized novel peptides (Hietter et al., 1989, 1990), and we recently identified three structurally-related, cysteine-rich, 8–9 kDa peptides. We present in this paper their complete structure determination. The amino acid sequence of these peptides is superimposable to that of neuroparsins isolated as dimers by Girardie et al., 1989. However, our experimental data lead us to propose that these molecules are monomers containing six intramolecular disulfide bridges.     

Intracellular Na+ and K+ contents of Zygosaccharomyces rouxii mutants defective in salt tolerance

Two of five Zygosaccharomyces rouxii mutants defective in salt tolerance, 152S (sat1) and 1717S (SAT3), were inviable in a nutrient medium (YPD) containing more than 1% NaCl. These two mutant cells contained significantly higher amounts of Na⁺ (298 μmol and 285 μmol per g cells of 152S and 1717S, respectively) but lower amounts of K⁺ (242 μmol and 176 μmol per g cells of 152S and 1717S, respectively) than three other mutants, 41S (sat2-1 [98 μmol Na⁺ and 326 μmol K⁺/g cells]), 197S (sat2-2 [103μmol Na⁺ and 336 μmol K⁺/g cells]), 1611S (SAT4 [139 μmol Na⁺ and 294 μmol K⁺/g cells]), as well as a wild-type strain, AN39 (61 μmol Na⁺ and 349 μmol K⁺/g cells), when cultured in YPD medium containing 0.8% NaCl. A KCl supplement, optimally 0.6 M, added to the medium somewhat restored the NaCl-hypersensitivity of 152S and 1717S with a concomitant decrease of intracellular Na⁺. This finding suggests that the NaCl-hypersensitive mutations are due to a defect in the Na⁺-regulating mechanism. The other three mutants showed weak responses to KCl in high NaCl-YPD. These five salt sensitive mutants and the wild-type strain retained the same levels of intracellular glycerol and arabitol when transferred into NaCl (5%)-YPD from YDP medium. This suggests that polyol accumulation is not the only mechanism of salt tolerance in Z. rouxii.     

Metabolism of 14C-indole-3-acetaldoxime by hypocotyls of Chinese cabbage

The metabolism of ¹⁴C-indole-3-acetaldoxime by Chinese cabbage hypocotyls was investigated using labelled tracer and incubation times less than 1 hr. Indole-3-acetonitrile, indole-3-methylglucosinolate and desulpho-indole-3-methylglucosinolate were the major metabolites, while IAA or other IAA precursors were not detected. The kinetics of the conversion of the aldoxime to the three metabolites was different under continuous feeding and pulse feeding conditions. The apparent K_m for the conversion of the aldoxime to the nitrile and the glucosinolate were 3.3 and 5.0 μM, respectively. Tissues of Isatis tinctoria, Helianthus annuus and Zea mays also formed significant amounts of the nitrile and Zea mays formed small amounts of indole-3-acetaldehyde.     

Genetic Regulation of Development in Sorghum bicolor: VI. The ma(3) Allele Results in Abnormal Phytochrome Physiology

Physiological processes controlled by phytochrome were examined in three near-isogenic genotypes of Sorghum bicolor, differing at the allele of the third maturity gene locus. Seedlings of 58M (ma₃^R ma₃^R) did not show phytochrome control of anthocyanin synthesis. In contrast, seedlings of 90M (ma₃ma₃) and 100M (Ma₃Ma₃) demonstrated reduced anthocyanin synthesis after treatment with far red and reversal of the far red effect by red. De-etiolation of 48-hour-old 90M and 100M dark-grown seedlings occurred with 48 hours of continuous red. Dark-grown 58M seedlings did not de-etiolate with continuous red treatment. Treatment of seedlings with gibberellic acid or tetcyclacis, a gibberellin synthesis inhibitor, did not alter anthocyanin synthesis. Levels of chlorophyll and anthocyanin were lower in light-grown 58M seedlings than in 90M and 100M. Etiolated seedlings of all three genotypes have similar amounts of photoreversible phytochrome. Crude protein extracts from etiolated seedlings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phytochrome was visualized with Pea-25, a monoclonal antibody directed to phytochrome from etiolated peas. The samples from all three genotypes contained approximately equivalent amounts of a prominent, immunostaining band at 126 kD. However, the sample from 58M did not show a fainter, secondary band at 123 kD that was present in 90M and 100M. The identity and importance of this secondary band at 123 kD is unknown. We propose that 58M is a phytochrome-related mutant that contains normal amounts of photoreversible phytochrome and normal phytochrome protein when grown in the dark.