Chemical polysialylation: Design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid–oxyntomodulin (PSA–OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA–OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA–OXM at 15 μmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.     

Optimal substrate profile for antibiotic fermentations

The optimal substrate profile is calculated by Pontryagin's maximum principle for a simplified mathematical model of microbial growth and product formation. This model is fitted to experimental data of turimycin fermentation. Sub optimal control is studied for comparison.     

Climatically induced interannual variability in aboveground production in forest-tundra and northern taiga of central Siberia

To investigate the variability of primary production of boreal forest ecosystems under the current climatic changes, we compared the dynamics of annual increments and productivity of the main components of plant community (trees, shrubs, mosses) at three sites in the north of Siberia (Russia). Annual radial growth of trees and shrubs was mostly defined by summer temperature regime (positive correlation), but climatic response of woody plants was species specific and depends on local conditions. Dynamics of annual increments of mosses were opposite to tree growth. The difference in climatic response of the different vegetation components of the forest ecosystems indicates that these components seem to be adapted to use climatic conditions during the short and severe northern summer, and decreasing in annual production of one component is usually combined with the increase of other component productivity. Average productivity in the northern forest ecosystems varies from 0.05 to 0.14 t ha⁻¹ year⁻¹ for trees, from 0.05 to 0.18 t ha⁻¹ year⁻¹ for shrubs and from 0.54 to 0.66 t ha⁻¹ year⁻¹ for mosses. Higher values of tree productivity combined with lower annual moss productivity were found in sites in northern taiga in comparison with forest-tundra. Different tendencies in the productivity of the dominant species from each vegetation level (trees, shrubs, mosses) were indicated for the last 10 years studied (1990–1999): while productivity of mosses is increasing, productivity of trees is decreasing, but there is no obvious trend in the productivity of shrubs. Our results show that in the long term, the main contribution to changes in annual biomass productivity in forest-tundra and northern taiga ecosystems under the predicted climatic changes will be determined by living ground cover.     

Conformational dynamics and pre-steady-state kinetics of DNA glycosylases

Results of investigations of E. coli DNA glycosylases using pre-steady-state kinetics are considered. Special attention is given to the connection of conformational changes in the interacting biomolecules with kinetic mechanisms of the enzymatic processes.     

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.     

High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1

Summary A defined medium was developed which, by means of a specific fed-batch mode, allows growth of the recombinant Escherichia coli strain TG1 (pBB210) up to a cell density of 60 g dry weight/l. Apart from glucose and aqueous ammonia fed as carbon and nitrogen sources, it was unnecessary to supply other nutrients or O₂-enriched air. Aqueous ammonia also served for pH control. The pO₂ level was kept at 20% saturation via closed-loop controls operating the two output variables of stirrer speed and glucose feeding rate. This fed-batch method prevented significant accumulation of acetate and other metabolic by-products. The recombinant E. coli expressed interferon alpha 1 more efficiently at a lower specific growth rate (_Pr 0.15 h^–1) than at the maximum specific growth rate (_max = 0.45 h^–1). Therefore, fermentation in the batch phase at _max was only allowed to continue up to a medium cell density. In the succeeding fed-batch phase, the specific growth rate was reduced to _Pr by increasing the stirrer speed according to an empirically developed time scale. Offprint requests to: D. Riesenberg     

The seasonal sensitivity of brown bear denning phenology in response to climatic variability

Background

For brown bears (Ursus arctos), hibernation is a critical part of the annual life cycle because energy savings during hibernation can be crucial for overwintering, and females give birth to cubs at that time. For hibernation to be a useful strategy, timing is critical. However, environmental conditions vary greatly, which might have a negative effect on the functionality of the evolved biological time-keeping. Here, we used a long-term dataset (69 years) on brown bear denning phenology recorded in 12 Russian protected areas and quantified the phenological responses to variation in temperature and snow depth. Previous studies analyzing the relationship between climate and denning behavior did not consider that the brown bear response to variation in climatic factors might vary through a period preceding den entry and exit. We hypothesized that there is a seasonal sensitivity pattern of bear denning phenology in response to variation in climatic conditions, such that the effect of climatic variability will be pronounced only when it occurs close to den exit and entry dates.

Results

We found that brown bears are most sensitive to climatic variations around the observed first den exit and last entry dates, such that an increase/decrease in temperature in the periods closer to the first den exit and last entry dates have a greater influence on the denning dates than in other periods.

Conclusions

Our study shows that climatic factors are modulating brown bear hibernation phenology and provide a further structuring of this modulation. The sensitivity of brown bears to changes in climatic factors during hibernation might affect their ability to cope with global climate change. Therefore, understanding these processes will be essential for informed management of biodiversity in a changing world.

     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.     

Zur Genetik der Myositis ossificans progressiva

Zusammenfassung Myositis ossificans progressiva ist durch ein bestimmtes Muster der Ossifikationen und der angeborenen Begleitmißbildungen an händen und Füßen gekennzeichnet. Zahnstellungsanomalien sind häufig, Infantilismus mit Hypogenitalismus bzw. verzögerte Genitalentwicklung kommen regelmäßig vor. Die Kranken sind im allgemeinen wegen der Verkrüppelung, der herabgesetzten Lebenserwartung und des Hypogenitalismus nicht fortpflanzungsfähig.Unter den rund 350 Fällen der Literature sind nur zweimal Vater und Sohn, zweimal Vater und Tochter und einmal — außer der Probandin — eine Tante mütterlicherseits betroffren. In weiteren 13 Fällen ist bei mindestens einem Familienmitglied eine Mißbildung der Gliedmaßen, davon siebenmal bei einem Elter, erwähnt.23 Probanden mit Myositis ossificans progressiva sind unsystematisch gesammelt worden. In ihren Familien kommen keine weiteren Fälle von Myositis ossificans vor. 37 von 46 Eltern und 33 von 55 Geschwistern konnten untersucht werden, dazu weitere 53 Familienangehörige. Von 28 Eltern liegen Röntgenaufnahmen der Hände und Füße vor. Die erhobenen Befunde sind uneinheitlich, und ein Zusammenhang mit Myositis ossificans ist fraglich. Allenfalls in zwei Fällen könnte man bei einem Elter den Verdacht auf eine Mutante haben: In einem Fall ist beim Vater seit der Kindheit der rechte Daumen im Grundgelenk versteift, die Endglieder beider Großzehen zeigen eine spornartige Epiphysenverbreiterung, und das linke Großzehenendglied ist etwas kürzer als das rechte; seine älteste Tochter hatte ein Myxosarkom am Metatarsophalangealgelenk der linken Großzehe. Im anderen Fall sind bei der Mutter in der Kindheit angeblich spontan kleine exostosenähnliche Auswüchse an der Stirn aufgetreten, und sie hat eine geringe Verkürzung der Grundphalanx der linken Großzehe.Über zwei konkordant betroffene EZ-Paare ist in der Literature berichtet worden.Chromosomenuntersuchungen an zwei Kranken von Viparelli und bei drei von unseren Kranken haben normale Karyogramme ergeben.Die Prüfung des Geburtenordnungseffektes an 23 selbst untersuchten und 45 Patienten der Literatur (nach dem Haldane-Smith-Verfahren) hat eine signifikant stärkere Besetzung der höheren Geburtenränge ergeben (Differenz zwischen Beobachtung und Erwartung von 6 A=339, dreifache Streuung des Erwartungswertes 3s=±226,2). Von den 68 Patienten stehen 39,7% an vierter oder höherer Stelle in ihrer Geschwisterreihe, während der entsprechende Anteil in der herangezogenen Vergleichsbevölkerung nur 23,6% beträgt.Das durchschnittliche Alter der Väter unserer 23 Patienten und weiterer 16 der Literatur beträgt 36,3 Jahre; es liegt um rund 4 Jahre höher als das Durchschnittsalter der Väter der Vergleichsbevölkerung.Die durchschnittliche Altersdifferenz der Elternpaare von 35 Patienten beträgt 6,2 Jahre. Sie liegt um etwa 2 1/2 Jahre höher als diejenige, die in der Durchschnittsbevölkerung angenommen werden kann. Die zunehmende Häufigkeit der Geburten von Kranken mit ansteigendem väterlichen Alter zeigt sich auch, wenn man in einzelnen Altersstufen den Prozentsatz von Vätern der Patienten in Bezug setzt zu den der Väter aller lebenden Neugeborenen einer angenommenen Durchschnittsbevölkerung (Tabelle 3). Der Anstieg der Geburtenkurve ist stärker als linear und ähnlich dem bei Achondroplasie und Apert-Syndrom.Myositis ossificans ist ein dominant erbliches Leiden, dessen Mutante einem starken Druck der Selektion unterliegt. Infolgedessen sind die Mehrzahl aller Fälle neue Mutanten, entstanden in den väterlichen Gonaden.

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Myositis ossificans progressiva is characterized by a distinct pattern of ossification and of accompanying congenital deformities of hands and feet. Anomalies of position of teeth are frequent, infantilism together with hypogenitalism, or delayed sexual development are found regularly. The patients usually have no children, as they are malformed, have a low expectation of life, and suffer from hypogenitalism.There are about 350 cases known in the literature. In two cases father and son were affected, twice father and daughter, and once a maternal aunt of a female proband. In 13 cases a deformity of extremities is mentioned in at least one member of the family, seven times in one parent.23 probands with myositis ossificans progressiva were collected and their families examined. It was possible to examine 37 of 46 parents and 33 of 55 siblings, and 53 other relatives. No more cases of myositis ossificans could be found. In 28 parents röntgenograms of the hands and feet were made. The results are not homogeneous and a relationship with myositis is doubtful. Only in two cases one could take into account the manifestation of a new mutant in one parent: In one case the right thumb of the father is stiffened in its metacarpo-phalangeal joint since childhood, the distal phalanges of both first toes show a spinelike broadening of the epiphysis, and the left distal phalanx of the first toe is a bit shorter than the right one. His eldest daughter had a myxosarcoma of the metatarso-phalangeal joint of the left first toe. In the other case exostose-like protuberances could be seen at the forehead of the mother, and the metatarso-phalangeal joint of the left first toe is a bit shorter than the other.Two monocygotic twin pairs are mentioned in the literature, both twins of each pair are affected.Chromosomes were examined in two patients by Viparelli and in three of our patients. All of them show a normal caryotype.The birth-order effect was examined in our 23 patients (see Table 2) and in 45 patients (see Table 4) of the literature (by the Haldane-Smith-method). Higher birth ranks were significantly more frequent (difference between observation and expectation of 6 A=339, 3-fold standard deviation of the expectation=±226,2).The average age of the fathers of our 23 patients and of further 16 patients of the literature is 36,3 years; it is about 4 years higher than the average age of the fathers of the control sample.The difference between mean ages of 35 parental pairs is 6,2 years. It is about 2 1/2 years higher than in the control sample. The increasing frequency of affected children with paternal age was examined by comparison of 5 years' gaps in a control sample (see Table 3). The slope of the age specific risk curve is steeper than linear and similar to that of achondroplasia, Apert-syndrome and haemophilia A (see Fig. 1).Myositis ossificans progressiva is caused by autosomal dominat mutations which are exposed to a strong selection pressure. Consequently the majority of all cases are new mutants, originating in the gonades of the fathers.

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Die Untersuchungen wurden mit Hilfe der Fritz Behrens-Stiftung durchgeführt.