Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.     

Paarungssystem und Fortpflanzungsstrategie der Beutelmeise (Remiz p. pendulinus)

Zusammenfassung Paarungssystem und Fortpflanzungsstrategie der Beutelmeise wurde in 10 Jahren in Süddeutschland und Österreich untersucht. Der Saisonablauf für und wird in einem Schema zusammengefaßt. Die Brutpflege übernimmt i. allg. nur ein Altvogel (bei ca. 60 % aller Brutnester , bei 10 % ). Unklar ist, wie die Abstimmung erfolgt, welcher Altvogel das Gelege betreut. Offenbar herrscht eine Konfliktsituation zwischen den Geschlechtern, die sich in zahlreichen Gelegeaufgaben niederschlägt (ca. 30 % aller Brutnester). Die Verhaltensweisen betreuen bzw. verlassen sind für und nicht gleichmäßig über die Brutsaison verteilt. Die Mediane für die Betreuung durch das und die Aufgabe der Nester liegen dicht beieinander, betreuen die Nester erst wesentlich später. In Illmitz/Österreich nahm die Gelegegröße mit fortschreitender Jahreszeit ab. Die Gelegegröße der aufgegebenen Gelege war kleiner als die zeitgleich zu erwartende Gelegegröße der betreuten Gelege. Wenn das abwandert, komplettiert es das Gelege offensichtlich nicht; damit wird für das zurückbleibende erfolgreiche Brutpflege möglich. Beide Geschlechter verfolgen unterschiedliche Strategien, um einen möglichst guten Fortpflanzungserfolg zu erreichen. Beim besteht die ganze Brutsaison hindurch Bereitschaft, mehrere Nester zu bauen und möglichst viele zu haben. Gegen Ende der Brutsaison, wenn nur noch wenige brutbereite verfügbar sind, übernehmen die u. U. selbst die Brutbetreuung. betreuen zu Beginn der Brutsaison komplette Gelege selbst. Bei rund 30 % der ist aber schon zu Beginn der Brutsaison die Bereitschaft vorhanden, das Gelege mit unvollständiger Gelegegröße nicht selbst zu betreuen und die Verantwortung hierfür dem zu überlassen. Zum Geschlechterkonflikt kommt es, wenn beide Strategien hinsichtlich Abwandern kollidieren; er läßt sich mit Hilfe der Spieltheorie erklären. Wegen der großen Wanderungen während der Brutperiode haben die Angaben zum Verpaarungsstatus der einzelnen Vögel Minimalcharakter. Mindestens 25 % aller und 12 % aller waren polygam. Daß während der ganzen Brutsaison keine Partner haben, ist nachgewiesen. Der Bruterfolg polygamer war signifikant größer als der von mit nur einem Brutnest. Polygame hatten größere Flügel als die übrigen; auch deutet sich an, daß alte erfolgreicher sind als junge. Eine Polygynierate von bis zu 60 % dürfte realistisch sein; die Polyandrierate dürfte bei ca. 30 % liegen. Polyandrisches Verhalten ist aber nicht immer mit einer Steigerung der Reproduktionsrate verbunden. Besonders zu Beginn der Brutsaison bedeutet die Abwanderung des mit hoher Wahrscheinlichkeit die Aufgabe des Nestes, da die in dieser Phase die Brutbetreuung in der Regel nicht übernehmen. Einzelne waren in der Lage, nacheinander zwei Bruten alleine großzuziehen.

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Mating system and strategy of reproduction in Penduline TitRemiz p. pendulinus

Summary From 1978 to 1987, 477 nests of the Penduline Tit were checked in Bavaria and Austria. Adult birds were marked individually with colour rings.In northern Bavaria, the major part of Penduline Tits started building nests in April and May (median date: May 10). As early as end of May a large number was leaving the breeding grounds. The population in the riverine forests of the river March/Austria basically followed the same cycle. On the other hand, birds breeding at Lake Neusiedl/Austria arrived as late as the beginning of June (median date for start of nestbuilding: June 23). A generally valid seasonal pattern for both sexes see fig. 1, 2. Penduline Tits exhibit uniparental care. Most of the nests (about 60 %) were attended by females, about 10 % by male and about 30 % were deserted, because neither the female nor the male decided to incubate (tab. 1).Early in the year, clutches were reared by the female or abandoned; preferably at the end of the breeding season males incubated. A significant relation between clutch size and season could be found at Illmitz/Austria. If average clutch size for female, male or no care are compared with the average clutch size of the respective five-day periods in which the medians of the three categories were found, a significantly smaller clutch size was apparent in abandoned nests. Due to extensive migratory behaviour during the breeding season, a calender effect of clutch size was not calculated for the Upper Main/Bavaria. However, clutch size in nests incubated by females exceeded the size of the abandoned ones. The incubation of smaller clutches by males in the same area is hard to explain. Insufficient data and/or the lack of potential mates as a consequence of early migration may account for it. If a female decides to abandon a nest she evidently leave the clutch incomplete, as if to give the male a commission to care for the young. Thereby, females economize on their reserves in order to be able to produce additional clutches with other males.To achieve optimal reproductive success both sexes follow different strategies. Males: During the whole breeding season males are able to construct a large number of nests to attract as many females as possible. At the end of the breeding season, when only few females are available, some of the males care for their broods themselves. Females: At the beginning of the breeding season, most females are ready to incubate complete clutches. But already at this time, approximately 30 % of the females desert incomplete clutches so as trying to give commission for brood care to the male.The battle of the sexes arises, if both sexes abandon the clutch. This conflict may probably be explained by the game theory. Due to the migrations during breeding season, an exact documentation of the mating status of individual birds was limited. Polygamy occured at least in 25 % of the males and 12 % of the females (tab. 4). A number of unpaired males was also evident during the whole breeding season. Polygynous males reached higher breeding success (tab. 8), and were larger (wing-length) as compared to the other males. Older males may be more successful than younger. According to the number of males leaving nests, the rate of polygyny might be up to 60 % and, due to the number of clutches abandoned by females, approx. 30 % of them are polyandrous. However, polyandrous behaviour was not linked to an increased reproduction rate. Especially at the beginning of the breeding season, most of the nests deserted by females were completely abandoned. Some females were able to raise two broods in one season.