Production of heterologous proteins in Bacillus subtilis: the effect of the joint between signal sequence and mature protein on yield

Summary We have previously made a set of DNA constructs by fusing the mature part of Bacillus licheniformis -amylase with the signal sequence of B. amyloliquefaciens -amylase at various distances from the signal sequence cleavage site. We observed that the level of -amylase production in B. subtilis depended strongly on the distance of the junction from the signal sequence cleavage site, with quite a sharp optimum distance. To test whether the effect is limited to the pair of -amylase signal sequence and mature protein, we analysed the protein production in a set of constructs in which an Escherichia coli \-lactamase was similarly joined at different distances from the -amylase signal sequence. Also in this case the distance seemed to be an important factor in affecting the level of production in B. subtilis. The observed effect might depend on the modulation of pre-protein folding, which in turn could affect the secretion level. Offsprint requests to: M. Sibakov     

Relations between planktivorous fish abundance,zooplankton and phytoplankton in three lakes of differing productivity

Hydrobiologia - Water chemistry, phytoplankton, zooplankton and fish populations werestudied over several years in three shallow, non-stratified lakes withdiffering nutrient loadings and fish...     

Glutathione depletion in isolated rat hepatocytes caused by styrene and the thermal degradation products of polystyrene

Depletion of reduced glutathione (GSH) was induced in isolated rat hepatocytes incubated with styrene or exposed for 120 min to products from oxidative thermal degradation of polystyrene. The depletion depended on the concentrations of styrene and on the degradation temperature. Styrene (1 mM) or products from degradation of polystyrene at 200°C (concentration of styrene in exposure atmosphere 0.7 ppm) had no detectable effect on glutathione levels in isolated hepatocytes. At higher degradation temperatures (250°C and 300°C, with styrene concentrations of 2.5 and 25 ppm, respectively) a rapid depletion was detected as well as with 3 mM styrene in incubation mixture. The latency of lactate dehydrogenase was affected neither by the polystyrene degradation products nor by the styrene added to the incubation mixture.     

Why do territorial male Tengmalm's owls fail to obtain a mate?

Non-breeding may occur because non-breeders are immature or somehow physiologically incapable of breeding, or because of a lack of resources (e.g. food resources, mating partners) needed to breed. There is, however, a lack of experimental evidence on whether bachelor males possessing territories and nest-sites are able to breed when supplemented with extra food or provided with mating partners. In vole-eating Tengmalm's owl, Aegolius funereus, we provided supplementary food and transferred females in nest-boxes of non-breeding males. Bachelor males that we supplemented with food did not attract mates at a higher frequency than unfed control males, which suggests that a lack of food did not influence the ability to attract a mating partner. In contrast, bachelor males presented with a female seemed to breed more frequently than bachelor males in the control group without mate addition. This suggests that scarcity of females may be an important reason for the high proportion of non-breeding males in the population (c. 25%) and excludes the possibility that non-breeding males are physiologically unable to breed. The operational sex ratio of the owl population at the time of mating may be male-biased, and some males may thus remain unpaired. Habitat and nest-box quality also seemed to be lower among bachelors than among breeding males. Received: 22 September 1997 / Accepted: 29 December 1997     

Muscle strength and history of heavy manual work among elderly trained women and randomly chosen sample population

The association between a history of heavy work and muscle strength was studied among 51 physically active women aged 66–85 years trained in sports and 41 women aged 70–81 years selected randomly from the population register. Maximal isometric muscle strength of hand grip, arm flexion, leg extension and trunk flexion and extension were measured using specially constructed dynamometers. The capacity of the abdominal muscles was evaluated by means of a sit-up test. The study included an interview dealing with the subjects' histories of heavy manual employment. The mean histories of heavy work for the trained and untrained women were 24 and 36 years, respectively. The trained women showed significantly greater maximal isometric muscle strength and abdominal muscle capacity than the untrained women. Among the trained women there was no correlation between the amount of heavy work and muscle performance. Among the untrained women the amount of heavy work correlated positively with maximal isometric trunk extension strength. The results would suggest that among elderly women, whether physically active or not, a history of heavy work has no systematic association with muscle strength.     

Age- and training-related changes in the collagen metabolism of rat skeletal muscle

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.     

Lipid content and utilization of lipids in planktonic copepods in Lake Pääjärvi,southern Finland

The variation of the lipid content of a planktonic copepod species, Eudiaptomus gracilis G. O. Sars, was studied in an oligotrophic lake, Pääjärvi, southern Finland, during 1982. The variation was caused by changes in the food resources and by the use of lipids for reproduction. The lipids are partly present in the form of droplets, which provide an energy reserve for some of food shortage, or may be used in reproduction. The utilization of lipid droplets for nutrition was studied by culturing specimens of two species, Eudiaptomus gracilis and Thermocyclops oithonoides G. O. Sars, in the absence of food. It was found that about half of the energy required for the 30-hour experimental period was obtained from the lipid droplets. However, not all the animal groups could use their lipid droplets and they had to use other reserves to satisfy their energy requirement. Lipid droplets seemed to have only a minor effect on the sinking rate of Eudiaptomus gracilis.     

Movement and disappearance of mountain birch defoliators are influenced by the interactive effects of plant architecture and induced resistance

Abstract. 1. Changes in herbivore movement and feeding behaviour may determine the efficacy of induced plant resistance by affecting the location of damage within the foliage and by modifying the vulnerability of herbivores to predators. 2. Observations of larval feeding sites were used to test whether induced resistance increased the movement of free‐living Epirrita autumnata Borkh. (Lepidoptera, Geometridae) larvae feeding on mountain birch [Betula pubescens ssp. czerepanovii (Orlova) Hämet‐Ahti]. The amount of defoliation at different canopy parts was measured to test the associated changes in the spread of damage within the foliage. 3. The architectural complexity of trees was measured to test its association with the disappearance of larvae from their hosts. The underlying hypothesis was that the architectural traits of the host plant could affect disappearance by influencing the frequency of herbivores encountering predators. 4. Distance between the consecutive feeding positions, the number of leaves damaged, and consumption of long shoot leaves all increased in trees with induced resistance. 5. Disappearance of larvae depended on the architectural complexity of trees. The effect of complexity differed between defoliation treatments, and may depend on the activity and number of predators in relation to the canopy size. 6. Accordingly, this study suggested that the interactive effects of plant architecture, induced resistance, and herbivore behaviour can determine the performance of herbivores on their host plant.     

Inference of Boolean Networks Using Sensitivity Regularization

The inference of genetic regulatory networks from global measurements of gene expressions is an important problem in computational biology. Recent studies suggest that such dynamical molecular systems are poised at a critical phase transition between an ordered and a disordered phase, affording the ability to balance stability and adaptability while coordinating complex macroscopic behavior. We investigate whether incorporating this dynamical system-wide property as an assumption in the inference process is beneficial in terms of reducing the inference error of the designed network. Using Boolean networks, for which there are well-defined notions of ordered, critical, and chaotic dynamical regimes as well as well-studied inference procedures, we analyze the expected inference error relative to deviations in the networks'' dynamical regimes from the assumption of criticality. We demonstrate that taking criticality into account via a penalty term in the inference procedure improves the accuracy of prediction both in terms of state transitions and network wiring, particularly for small sample sizes.     

Utilization of intrachain 4'-C-azidomethylthymidine for preparation of oligodeoxyribonucleotide conjugates by click chemistry in solution and on a solid support

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.