Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Occurrence of Nitrate Reductase and Molybdopterin in Xanthomonas maltophilia

Fifteen of 23 ATCC strains and 2 of 9 clinical isolates of Xanthomonas maltophilia, all of which grew aerobically on ammonia, but not nitrate, as a sole nitrogen source, reduced nitrate to nitrite. X. maltophilia failed to grow anaerobically on complex medium with or without nitrate, so it is considered an obligate aerobe. Nitrate-reducing strains contained reduced methyl viologen nitrate reductase (MVH-NR) with specific activities ranging from 49.2 to 192 U mg of protein⁻¹. Strain ATCC 17666 doubled its cell mass after 3 h of growth on nitrate broth under low aeration, possessed maximal MVH-NR activity, and converted the added nitrate to nitrite, which accumulated. Dissolved oxygen above 15% saturation greatly suppressed nitrite formation. All strains, except ATCC 14535, possessed between 0.25 and 5.05 pmol of molybdopterin mg of protein⁻¹ as measured by the Neurospora crassa nit-1 assay. The molybdopterin activity in the soluble fraction sedimented as a single symmetrical peak with an s_20,w of 5.1. Studies identified MVH-NR in selected strains as a membrane-bound protein. The deoxycholate-solubilized MVH-NR sedimented as a single peak in sucrose density gradients with an s_20,w of 8.8. The MVH-NR of X. maltophilia has the physical characteristics of a respiratory nitrate reductase and may enable cells to use nitrate as an electron sink under semiaerobic conditions.     

Effects of Azadirachtin on reproduction in the African armyworm (Spodoptera exempta)

Azadirachtin, applied topically to final instar larvae of the African armyworm (Spodoptera exempta, Walker) (Lepidoptera: Noctuidae), adversely affected oogenesis and reproductive maturation in subsequent female moths. Moths obtained from such treated larvae failed to mature their oocytes, probably as a result of interference of azadirachtin with vitellogenin synthesis and/or its uptake by developing oocytes. Such larval treatment also caused substantial decreases in fecundity and although fertility in affected females was not decreased significantly, emerging larvae were less viable, less than 40% reaching the fourth instar.Closer examination revealed that protein levels as well as fat body development in female moths were suppressed by azadirachtin. Prospects for field control of this pest with neem are discussed in the light of these findings.

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Résumé L'application locale d'azadirachtine sur des chenilles de dernier stade de S. exempta Walker nuit à l'ovogenèse et à la maturation des ovaires des futures femelles. Les papillons provenant des chenilles traitées ne produisent pas d'ovocytes mûrs, probablement par suite de l'interférence de l'azadirachtine avec la synthèse de la vitellogénine et/ou son incorporation dans les ovocytes en développement. De tels traitements larvaires provoquent aussi une diminution importante de la fécondité et bien que la fertilité des femelles touchées ne diminue pas significativement, les chenilles néonates sont moins viables, moins de 40% atteignant le 4^e stade. Un examen plus précis a révélé que la teneur en protéines et le développement du corps gras des papillons femelles étaient altérés par l'azadirachtine. Des perspectives d'utilisation du NEEM dans la lutte contre cet insecte ont été discutées à la lumière de ces résultats.

     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

The influence of noradrenaline on the process of protein/peptide secretion in the mammalian pineal organ

Summary From studies conducted with the pineal organ of the mouse, it was ascertained that for the in vitro investigation of secretory processes (synthesis and release) of proteic/peptidic compound(s), a culture time of 5 to 14 days is optimal. A 5-day organ culture was therefore chosen to study the effects of noradrenaline on these secretory processes.Addition of noradrenaline to the culture medium provokes, in pineal explants of the normal mouse and the eyeless mouse, an inhibition of the secretory process, characterized by the formation of granular vesicles. In the hamster and rat, however, opposite results were obtained. Moreover, it appears that noradrenaline, at least in the rat, may also be involved in the regulation of the ependymal-like secretory process.The present results indicate clearly that noradrenaline (thus, the sympathetic innervation) is implicated in the regulation of the production of proteic/peptidic hormonal agents, but that the effect of this neurotransmitter is species-specific. This could explain the numerous contradictory results reported in the literature.IBRO/UNESCO fellow     

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

The influences of buffers and iron chelators on the rate of autoxidation of Fe²⁺ were examined in the pH range 6.0–7.4. The catalysis by Fe²⁺ and Fe³⁺ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe²⁺, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30°C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe³⁺ over Fe²⁺, Fe²⁺ autoxidizes rapidly in the pH range 6.0–7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe²⁺ stabilize both Fe²⁺ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe³⁺ in non-chelating buffers or in the presence of strong chelators of Fe²⁺, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe²⁺ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe²⁺ and Fe³⁺. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe²⁺ or both dithiothreitol and iron.     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Adoptive chemoimmunotherapy of a syngeneic murine lymphoma with long-term lymphoid cell lines expanded in T cell growth factor

Summary Recently techniques have been developed for the long-term growth of cytotoxic T-lymphoid cells in vitro with T cell growth factor (TCGF). We have investigated the use of these in vitro-expanded T cells for the immunotherapy of a disseminated syngeneic murine FBL-3 lymphoma. In this model, mice with disseminated tumor were treated on day 5 with 180 mg cytoxan/kg and then 5 h later were given lymphoid cells IP. In vivo-immunized lymphocytes resulted in significantly improved survival in three of three experiments, curing 52% of 38 animals, compared with treatment with cytoxan alone (0 of 31 cured) or cytoxan plus unimmunized cells (0 of 40 cured) (P<0.0005). In vivo-immunized lymphocytes were re-exposed to FBL-3 tumor in vitro for 5 days in complete medium (CM) or lectin-free TCGF (LF-TCGF). Both groups showed significantly improved survival in six of six experiments. Cytoxan cured 17% of 66 animals, while cytoxan plus normal lymphocytes after IVS cured 6% of 47 animals. In vivo-immunized cells resensitized in vitro to FBL-3 in CM or LF-TCGF cured 82% of 50 animals (P<0.001) and 72% of 61 animals (P<0.001), respectively. Cells from in vivo- and in vitro-sensitized lymphocytes exhibited no cytotoxicity in our in vitro ⁵¹Cr-release assay; expansion of these cells resulted in significant specific lysis of fresh FBL-3 targets. Adoptive transfer of immune lymphocytes resensitized to FBL-3 tumor in vitro and expanded in LF-TCGF conferred a significant survival benefit (P<0.001, curing 7 of 27 animals) compared with all controls. These expanded cells were then continuously grown in LF-TCGF for 2 1/2 months. Again, in vivo-immunized lymphocytes resensitized to FBL-3 tumor and expanded in LF-TCGF for 2 1/2 months cured 56% of the animals with disseminated tumor, significantly prolonging survival over that recorded in any control group (P<0.0002). Irradiation of these same cells totally abolished their efficacy. Clones were generated from IVS and continuously grown in LF-TCGF. Two of these clones were very cytotoxic for fresh FBL-3 (>4,000 lytic units/10⁶ cells). When adoptively transferred to mice in this chemoimmunotherapy model these cytotoxic clones significantly enhanced survival over that recorded following treatment with cytoxan alone (P<0.00001), though prolongation of survival was small. Implications of these results for application of these techniques to other less antigenic tumors and human cancers are discussed.