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.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

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.     

Einfluß der Konzentration der Zuckerlösung auf den Gesang und das Balzverhalten des Gelbbauchnektarvogels (Nectarinia venusta)*

Different concentrations of a sucrose solution vary the courtship song and behaviour of the male yellow-bellied sunbird Nectarinia venusta- the duration of subsong, total singing duration, and the absolute number of full song phrases. With high concentrations the sunbird sings more full song phrases but less subsong during the courtship season than otherwise. The various effects are described.     

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     

Substance P-like immunoreactivity in the parietal eye visual system of the lizard Uta stansburiana

Summary We examined the parietal eye visual system of the iguanid lizard Uta stansburiana for the presence of substance P-like immunoreactivity by use of both immunofluorescence and peroxidase-antiperoxidase techniques. In the parietal eye no substance P-containing somata were found; however, its plexiform layer contained small (ca. 1 m diam) immunoreactive fibers. These fibers apparently originate outside the parietal eye. Immunoreactive fibers also were found in the parietal nerve, the dorsal sac, and the leptomeninx of the pineal gland. No labeled somata were observed in any of these regions in either normal or colchicine treated animals. Previously we demonstrated that a system of centrifugal fibers to the parietal eye originates from neurons in the dorsal sac (Engbretson et al. 1981). The apparent absence of substance P-containing neurons in the dorsal sac suggests that the substance P-containing fibers in the parietal eye are not the previously observed centrifugal fibers. The source of the substance P-containing fibers in the parietal eye is unknown. The pars dorsolateralis of the left medial habenular nucleus receives a dense substance P-positive projection. No such projection was seen in the right habenula. Simultaneous visualization of the terminals of ganglion cells of the parietal eye (labeled with orthograde intraaxonally transported horseradish peroxidase) and substance P-like immunofluorescence showed that the locus of habenular immunoreactivity is distinct from the projection field of the parietal eye. Thus the substance P-positive terminals in the habenula do not originate in the parietal eye. Transection of the parietal nerve confirmed this conclusion.     

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.