The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis)

Background

We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies.

Methodology/Principal Findings

A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases.

Conclusions/Significance

The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.     

The eukaryotic plasma membrane as a nutrient-sensing device

In eukaryotic cells, G-protein-coupled receptors (GPCRs), non-transporting nutrient carrier homologues and active nutrient carriers have been recently shown to function as sensors that directly monitor the level of nutrients in the extracellular environment. The plasma membrane is not only the cellular boundary at which signalling molecules that govern metabolism and proliferation are detected, but also the boundary across which nutrients that sustain the generation of energy and building blocks are transported. Nutrient sensors combine these functions in various ways. Classical receptor proteins detect the presence of nutrients, carriers combine the functions of nutrient transporters and receptors, and carrier homologues have lost their transport capacity and become pure receptors. The activation of signal transduction pathways by nutrients adds a new layer to the regulatory network that controls metabolism and proliferation. Nutrient sensors highlight the importance of both nutrients as signalling molecules and nutrient carriers as receptors for signalling pathways.     

Apple polygalacturonase inhibiting protein1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins

Extracts from apple fruit (cultivar "Granny Smith") inhibited the cell-wall degrading polygalacturonase (PG) activity of Colletotrichum lupini, the causal agent of anthracnose on lupins, as well as Aspergillus niger PG. Southern blot analysis indicated that this cultivar of apple has a small gene family of polygalacturonase inhibiting proteins (pgips), and therefore heterologous expression in transgenic tobacco was used to identify the specific gene product responsible for the inhibitory activity. A previously isolated pgip gene, termed Mdpgip1, was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The mature MdPGIP1 protein was purified to apparent homogeneity from tobacco leaves by high salt extraction, clarification by DEAE-Sepharose and cation exchange HPLC. Purified MdPGIP1 inhibited PGs from C. lupini and PGs from two economically important pathogens of apple trees, Botryosphaeria obtusa and Diaporthe ambigua. It did not inhibit the A. niger PG, which was in contrast to the apple fruit extract used in this study. We conclude that there are at least two active PGIPs expressed in apple, which differ in their charge properties and ability to inhibit A. niger PG.     

Psychosocial functioning, self-perception and body image and their auxologic correlates in growth hormone and oestrogen-treated young adult women with Turner syndrome

BACKGROUND: Few data are available on the psychosocial status of growth hormone (GH) and oestrogen treated women with Turner syndrome (TS). In this study, we evaluated psychosocial functioning, self-concept and body image in GH and oestrogen treated young adult women with TS and we studied the relationship with auxological parameters. PATIENTS AND METHODS: Thirty women with TS (mean +/- SD age: 22.1 +/- 2.4 years), all treated with GH and oestrogens if indicated, and an age-matched reference group of 44 non-Turner female students (age: 20.5 +/- 2.1 years) completed 3 questionnaires evaluating, respectively, behavioural and emotional problems (Young Adult Self Report), self-concept (Self Perception Profile for College Students) and body-image (Body Attitude Scale). RESULTS: TS patients did not report more behavioural and emotional problems compared to the non-TS females except for attention problems; they even reported fewer problems on some subscales (somatic complaints, thought problems, delinquent behaviour). TS patients did not differ from the non-TS female group in their bodily satisfaction. TS patients, particularly patients with a 45,X karyotype, perceived themselves as less socially competent. BMI was significantly related to the appraisal score of the Body Attitude Scale, whereas height was not related to any of the evaluated psychosocial parameters. CONCLUSION: The psychosocial adaptation of young adult women with TS, diagnosed at an early age and treated during childhood with GH and oestrogens if indicated, appears to be quite satisfactory. Follow-up of adult TS patients should not neglect the problem of overweight and associated psychosocial consequences.     

Anpassungen der H?moglobine von Streifengans (Anser indicus), Andengans (Chloephaga melanoptera) und Sperbergeier (Gyps rueppellii) an hypoxische Bedingungen

Zusammenfassung Das Phänomen ungewöhnlicher Hypoxietoleranz bei Vögeln wird auf der Basis von Struktur-Funktionsbeziehungen der Hämoglobine dargestellt. Die Hämoglobine der Streifengans (Anser indicus), der Andengans (Chloephaga melanoptera) und des Sperbergeiers (Gyps rueppellii) wurden untersucht. Dabei zeigt sich, daß die Hypoxietoleranz auf jeweils eine definierte Aminosäuresubstitution zurückzuführen ist, welche die Wechselwirkung zwischen den Untereinheiten des Hämoglobins schwächt. Dadurch ergeben sich für die einzelnen Hämoglobine unterschiedliche Affinitäten zum Sauerstoff. Für die Streifengans (Austausch 119 Pro Ala) und den Sperbergeier (Austausch 34 Thr/Ile; multiple Hämoglobine) entsteht eine zweibzw. dreifache Kaskade von Hämoglobinen unterschiedlicher Sauerstoffaffinität, für die Andengans (Austausch 55 Leu Ser) eine erhöhte intrinsische Affinität des Gesamtblutes. Diese beiden molekularen Muster entsprechen der Unterscheidung zwischen transitorischer und permanenter Hypoxietoleranz. Auffällig ist, daß die Sauerstoffaffinität der Hämoglobine der Andengans und der Streifengans über die gleiche Wechselwirkung zwischen den Untereinheiten reguliert werden; die Mutationen liegen jedoch auf verschiedenen Globinketten.

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Adaptation of the hemoglobins of Barheaded Goose (Anser indicus), Andean Goose (Chloephaga melanoptera) and Rüppell's Griffon (Gyps rueppellii) to life under hypoxic conditions

Summary The phenomenon of unusual hypoxic tolerance of birds is elucidated on the basis of structural and functional relationships of hemoglobins. Hemoglobins of Barheaded Goose (Anser indicus) flying over the Himalayan mountains at 8848 m, of Andean Goose (Chloephaga melanoptera) living at 6000 m in the Andes and of Rüppell's Griffon (Gyps rueppellii) reaching altitudes of 11 300 m were investigated. Hypoxic tolerance turned out to be the result of clearcut amino-acid substitutions weakening the interaction of the subunits of the hemoglobin. This entails different affinities of the single hemoglobins. For the Barheaded Goose (mutation 119 Pro Ala) and the Rüppell's Griffon (mutation 34 Thr/Ile; multiple hemoglobins) there is a two-stage resp. a three-stage cascade of hemoglobins of graded oxygen affinities, for Andean Goose (mutation 55 Leu Ser) the affinity of whole blood is raised. These two molecular patterns correspond to the distinction between transitory and permanent hypoxic stress. It is worth mentioning that the oxygen affinities of the hemoglobins of Barheaded Goose and Andean Goose are regulated via the same interface of the molecule; the substitutions, however, are found on different globin chains.

     

Estimation of the biofilm formation ability of Pseudomonas putida in discrete samples from continuous culture

Summary Test systems were set up in order to evaluate the ability of biomass from a continuous culture to form biofilms. A film-forming strain of Pseudomonas putida was used as the test organism. The adsorption of resting cells onto glass surfaces was measured in specially designed chambers containing 1 ml of cell suspension. Both the quantity and the physiological activity of the adsorbed cells, in terms of optical density after detachment and pH change of a substrate exposed to the adsorbed cells, were measured. The analysis of biomass from continuous cultures of Pseudomonas putida verified the suitability of the methods. Furthermore, other properties of importance to biofilm formation such as hydrophobicity and flocculation capacity of the cells were investigated.It was shown for samples deriving from different dilution rates that the cell adsorption rate drastically increased at dilution rates higher than the _max of the culture. Simultaneously, higher values of hydrophobicity and flocculation capacity were observed.It was also shown that the age and thickness of the biofilm subsequently produced in the continuous culture influenced the metabolic activity per unit of biomass attached to the surface. The methods described in this investigation may facilitate the study of parameters important to biofilm formation as well as the metabolic activity of the attached biomass.     

Vinblastine-induced precipitation of phloem proteinsin vitro

Summary Purified proteins from sieve tubes ofCucurbita maxima were precipitated with vinblastine and the precipitates were analyzed with the electron microscope. Filaments (35–40 Å in diameter) and tubular structures (160 Å in width) were observed in negatively stained preparations. The predominant structures of the negatively stained and of the thin sectioned material, however, were membrane-like sheets (100–120 Å in width) which showed the typical unit membrane aspect.Dedicated to Professor Dr. W.Schumacher on his 70th birthday.The investigations were supported by the Deutsche Forschungsgemeinschaft.     

Zur Herkunft und Struktur der Plasmafilamente in Assimilatleitbahnen

Zusammenfassung Für die den Stoffleitbahnen eigenen fädigen Strukturen wird die Bezeichnung Plasmafilamente vorgeschlagen.Wie erste Untersuchungen zur Differenzierung der Siebelemente zeigen, entstehen die Plasmafilamente bei Dioscorea direkt im Cytoplasma. Ihre Verbreitungsweise und ihr Feinbau in frühesten Differenzierungsstadien lassen vermuten, daß sie unmittelbar aus dem Grundplasma hervorgehen. Das Netzwerk der Plasmafilamente ist stets durchsetzt von Elementen des ER und zu keiner Zeit von einer Membran umgeben.Das Einzelfilament hat bei Dioscorea, Primula, Cuscuta und Cucumis eine unbestimmte Länge und einen Durchmesser von 120–150 Å. Im Querschnitt zeigt es den Aufbau aus einem osmiophilen Ring mit einem elektronenlichten Binnenraum. Dem entsprechen in der Längsaufsicht zwei äußere kontrastreiche und eine innere kontrastarme Schicht.Der Aussagewert der Filamentfeinstruktur wird unter Berücksichtigung ähnlicher Strukturen und ihrer Bedeutungen in der tierischen und pflanzlichen Zelle diskutiert.

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On the plasmatic filaments in assimilate conducting cells, their development and fine structure

Summary Taking into account the literature on the so-called sieve-tube slime (mictoplasm, slime strands) and regarding its fine structure more in detail the term plasmatic filament (Plasmafilament) is proposed and will be used in this paper to characterize the individual exceedingly fine subunit of the plasmatic network (or slime) in sieve elements. Up to now plasmatic filaments have mostly been erroneously called fibrils. The dimension of a fibrill has now been defined anew and differentiated from its subunit plasmatic filament.In the first part of these investigations some aspects of the development of plasmatic filaments and their spreading over the total lumen of Dioscorea sieve elements will be reported.Previous to the first appearance of filaments the later sieve element abounds in plasmatic components, the groundplasm being extremely rich in ribosomes (Fig. 1). The difference between young sieve elements and the neighbouring parenchyma cells is nearly imperceptible apart from a slight variation in ribosome density. Plastids are very useful in distinguishing these two cell types from each other. The development of osmiophilic inclusions that characterize sieve-element plastids in Dioscorea has already been initiated in these very young cells.The earliest stages in the formation of plasmatic filaments that up to now have been revealed in Dioscorea show masses of filaments, some short and granular in appearance (Fig. 2: *), some already elngated and filamentous (Fig. 2: F). After expanding over the entire cell those filaments still look like having their origin directly in groundplasm (Fig. 5). Elements of the ER-system and many ribosomes cross the plasmatic filaments during all developmental stages of their network, which is at no time surrounded by any membrane.In sieve elements of Dioscorea, Primula, Cuscuta and Cucumis our investigations furthermore yielded some detail on the filament substructure. A cross-sectioned plasmatic filament is composed of an osmiophilic outer ring with a light centre (Fig. 11) corresponding in a longitudinal view to two deeply contrasted outer layers and an inner one without any contrast (Fig. 8). An individual filament has an overall diameter of 120–150 Å and an up to now indeterminable length that exceeds at least several microns.The real nature of these fine structures will be discussed in relation to similar structures and their meaning in plant and animal cells.

     

Urinary Pyrophosphate in Normal Subjects and in Stone Formers

The urinary excretion of inorganic pyrophosphate was determined in nine normal subjects and also in eight patients with recurrent calcium-containing renal stones during varied levels of phosphate intake. The excretion of pyrophosphate and orthophosphate is virtually the same in the two groups at all levels of phosphate intake. It appears unlikely that a consistently reduced urinary excretion of pyrophosphate is a factor in the formation of urinary calculi. Pyrophosphate excretion rose and calcium excretion fell with increasing phosphate intake; this might be expected to have a beneficial effect in patients with recurrent calcium stones.