Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development.

To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 (A. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development.     

Coexistence of Banksia species in southwestern Australia: the role of regional and local processes

Abstract. 60 of the 75 Banksia species are confined to southwestern Australia where five or six species often coexist. We explored the role of regional species richness, niche differentiation, and habitat specialization in structuring banksia assemblages. The diversity of growth forms and categories of seed production and response to fire were assessed in actual assemblages at 40 sites throughout southwestern Australia. Diversity indices at each site were compared with those from null communities assembled on the basis of the abundance and sociability of taxa in regional species pools. The relationship between local and regional species richness suggests that processes at the scale of 100-m² quadrats limit local richness and therefore coexistence. However, there was no consistent evidence that taxa are differentiated by growth form or regeneration strategy. No particular biological profile makes a banksia adept at coexisting with a wide range of other taxa. Habitat specialization is an important factor contributing to lower local richness than would be predicted from niche differentiation of taxa in regional pools. There is recent empirical evidence of several mechanisms whereby the number of coexisting banksias is increased beyond the limits suggested by simple niche theories. Variability in the fire regime also provides a mechanism for maintaining local species richness because different fires favour recruitment of different taxa.     

A shared epitope found in the major paralysis inducing tick species of Africa

Cross-reactivity between all the paralysis inducing tick species of veterinary relevance in Africa was demonstrated, by using a monoclonal antibody directed against the paralysis inducing toxin of Rhipicephalus evertsi evertsi. Western blot results, together with amino acid composition studies indicated that this monoclonal antibody recognizes protein bands of similar molecular mass and amino acid composition in R. evertsi evertsi and Ixodes rubicundus. This suggests that the Karoo paralysis toxin of I. rubicundus is possibly also a trimer with a high degree of homology to the spring lamb paralysis toxin of R. evertsi evertsi. The conclusive identity of these protein bands of 1. rubicundus could not be shown. Bio-assay studies performed on 1-day-old chickens suggested that the anti-spring lamb paralysis toxin monoclonal antibody also recognizes the paralysis toxin present in Argas (Persicargas) walkerae, by rendering some degree of protection against the effect of this toxin.     

Metabolism of dodecyldimethylamine by Pseudomonas MA3

Pseudomonas MA3 was isolated from activated sludge on the basis of its capacity to use dodecyldimethylamine as a sole carbon (C) and energy source. Dodecylamine, dodecanal, dodecanoic acid and acetic acid also supported growth of Pseudomonas MA3. Dodecyldimethylamine-grown cells oxidized a wide range of alkylamine derivatives, dodecanal, dodecanoic acid and acetic acid. Degradation of the alkyl chain of dodecyldimethylamine by Pseudomonas MA3 appeared from the stoichiometric liberation of dimethylamine. A dehydrogenase catalysed the cleavage of the C_alkyl-N bond. The first intermediate of the proposed degradation pathway, dodecanal, accumulated in the presence of decanal used as a competitive inhibitor. The second intermediate,dodecanoic acid, was formed in the presence of acrylic acid during the degradation of dodecyldimethylamine. Dodecanal was converted into dodecanoic acid by a dehydrogenase and dodecanoic acid was then degraded via the oxidation pathway.     

Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus

The production of extracellular inulinase (\-1,2-d-fructan fructanohydrolase, EC 3.2.1.7) was studied in fed-batch cultures of the yeast Kluyveromyces marxianus CBS 6556 at 30 and at 40° C. At both temperatures, the final biomass concentration exceeded 100 g·l^–1 and more than 2 g enzyme. L^–1 of culture supernatant was produced. The biomass yield on O₂ at 40° C was substantially lower than at 30°C. Nevertheless, at 40° C a growth rate of 0.20 h^–1 could be maintained for a longer period than at 30° C. The unexpected higher O₂-transfer rate at 40°C is probably due to a lower viscosity of the culture broth. The 40°C fermentation took only 33 h as compared to 42 h at 30° C. These results indicate that K. marxianus is a promising host for the extracellular production of heterologous proteins under the control of the inulinase promoter.     

Cometabolic degradation of chloroallyl alcohols in batch and continuous cultures

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.