Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

Background

We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.

Results

K14 IBs were electroporated into SW13 cells grown in culture together with a ??reporter?? plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.

Conclusions

Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.     

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

Background  

In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.     

Airborne pollen of Betula,Corylus and Alnus in Zagreb,Croatia. A three‐year record

The aim of this study was to construct a picture of the influence of meteorological conditions on the start and duration of the airborne Betulaceae pollen season and the pollen concentrations in the atmosphere of Zagreb, Croatia. The study during three seasons (2002–2004) used a 7‐day Hirst‐type volumetric pollen and spore trap. Total annual airborne pollen of Alnus, Corylus and Betula greatly varied from year to year. The differences in the dates of onset of airborne pollen presence of Alnus, Corylus and Betula noted in Zagreb in 2002–2004 were controlled by weather conditions, particularly temperature and precipitation. In all years studied, airborne pollen peaks were recorded on days with temperature above 0°C and without or minimal precipitation. The mean number of days with airborne pollen concentrations exceeding levels which provoke symptoms of an allergic reaction was 15, 16 and 29 days for alder, hazel and birch, respectively. The results of the present study may provide useful data for allergologists to reach accurate diagnoses, and timely information on concentrations of airborne pollen types and concentrations for individuals with pollen hypersensitivity.     

Seasonal, daily and intradiurnal variation of PM10, NO2, NO and O3 in residential part of Zagreb, Croatia

Pollutants such as particulate matter, nitrogen oxides, carbon oxides, ground-level ozone, etc. are harmful to human health. Study of pollutant variation and its relationship with both dynamic and thermodynamic atmospheric boundary layer (ABL) structures is of importance not only for environmental protection but also for the public at large. The aim of this study was to analyze seasonal, daily and intradiurnal variation of PM10, NO2, NO and O3 in a residential part of an urban area, and the effect of some meteorological parameters. The study was conducted from January 1 till December 31, 2004 in the City of Zagreb using following methods: beta radiation absorption, chemiluminescence and UV photometry. The results presented in this article, show the dependence of air pollution levels upon traffic density, seasons and meteorological conditions. Considering the level of air pollution relative to the regulated limit and tolerated values, the measured 24-hour concentrations of all study pollutants exceeded the borderline values and/or tolerated values, however, the number of days with such pollutant concentrations did not exceed the allowed frequency. This is a preliminary study with the main objectives to point to the possible identification of the source of pollution and to assess the level of air contamination according to the new national legislation coordinated with European regulations. Future measurements and studies should evaluate in detail the causes of the concentration levels detected.     

Diurnal variation in airborne pollen concentrations of the selected taxa in Zagreb, Croatia

The number of individuals allergic to plant pollen has recently been on a constant increase. The knowledge of diurnal distribution and abundance of allergenic pollen types, their patterns and response to source position and weather is useful to correlate hay fever symptoms with the presence of allergenic pollen in the atmosphere. The aim of this study was to determine diurnal distribution of total airborne pollen, pollen of particular allergenic taxa, possible variation in diurnal pollen distribution at measuring sites placed at different heights, and effect of some meteorological parameters on airborne pollen concentrations. A 7-day Hirst-type volumetric pollen trap was used for pollen sampling. Qualitative and quantitative pollen analysis was performed under a light microscope (magnification x400). Total pollen of all plant taxa (Ambrosia sp., Betula sp., Cupressaceae, Urticaceae, Poaceae, Quercus sp., Fraxinus sp., Alnus sp., Corylus sp., Populus sp., Pinus sp., Picea sp.) observed showed a regular diurnal distribution at both sampling sites in both study years, with a rise in the pollen concentration recorded after 4.00 a.m. and 6.00 a.m., respectively. The peak pollen concentration occurred between 12.00 a.m. and 4.00 p.m., and the lowest diurnal pollen concentrations were recorded overnight. About 50% of the 24-h pollen concentration were released to the atmosphere between 10.00 a.m. and 4.00 p.m. The timing and size of diurnal peaks were closely related to high temperature, low humidity and south-west maximum wind direction.     

Dorsal longitudinal stretch receptor of Drosophila melanogaster larva - fine structure and maturation

Insects possess two types of sensory neurons: ciliated type I sensory neurons that innervate external sensory organs and chordotonal organs, and type II sensory neurons that form a subepidermal plexus or innervate stretch receptors. Among stretch receptors, a dorsel longitudinal stretch receptor is highly conserved in insects, being found in all insect orders investigated. Here we describe the topology and anatomical structure of this receptor in the fruit fly embryo and larva using transmission electron microscopy and single cell staining for fluorescence microscopy. The receptor is composed of the dorsal bipolar dendrite neuron, which arises from an archetypal cell lineage, its sister glial cell and the peripheral glial cell accompanying the nerve. The neuron is situated among the muscles in the dorsal body wall on the intersegmental nerve. Its two dendrites stretch the length of the segment to the segmental folds. The neuron is wrapped by both glial cells and surrounded by a common basal lamina, which fans out at the dendritic tips to attach them to the epidermal cells at the segmental borders.     

Airborne ragweed (Ambrosia artemisiifolia L.) pollen concentrations in Croatia, 2002–2004

The aim of this study was to determine the onset, length and end of the ragweed pollen season, taking into account diurnal, day-to-day, monthly and annual pollen variations, the effect of some meteorological parameters on atmospheric pollen concentrations and possible differences in the airborne pollen season and concentration due to sampling site. Airborne pollen was collected at three sites in central Croatia (Zagreb, Samobor and Ivanić Grad) during three pollen seasons (2002–2004). Seven-day Hirst-type volumetric pollen traps were used for pollen sampling. Ragweed pollen was the third most abundant pollen type to occur in the atmosphere of central Croatia. Total Ambrosia pollen concentration was the highest in the 2003 pollen season and the lowest in 2004 at all sampling sites. Maximum emissions were restricted to August and September. Intradiurnal periodicity showed a peak from 1000 to 1200 hours. The concentration of ragweed pollen during the pollen season was greatly influenced by temperature and precipitation: on rainy days accompanied by temperature decline, the air pollen concentration decreased abruptly. The results of this study are aimed at helping to alleviate the symptoms of allergic reactions in individuals with ragweed pollen hypersensitivity, thus improving their quality of life.     

Adaptive Control of Exoskeleton Robots for Periodic Assistive Behaviours Based on EMG Feedback Minimisation

In this paper we propose an exoskeleton control method for adaptive learning of assistive joint torque profiles in periodic tasks. We use human muscle activity as feedback to adapt the assistive joint torque behaviour in a way that the muscle activity is minimised. The user can then relax while the exoskeleton takes over the task execution. If the task is altered and the existing assistive behaviour becomes inadequate, the exoskeleton gradually adapts to the new task execution so that the increased muscle activity caused by the new desired task can be reduced. The advantage of the proposed method is that it does not require biomechanical or dynamical models. Our proposed learning system uses Dynamical Movement Primitives (DMPs) as a trajectory generator and parameters of DMPs are modulated using Locally Weighted Regression. Then, the learning system is combined with adaptive oscillators that determine the phase and frequency of motion according to measured Electromyography (EMG) signals. We tested the method with real robot experiments where subjects wearing an elbow exoskeleton had to move an object of an unknown mass according to a predefined reference motion. We further evaluated the proposed approach on a whole-arm exoskeleton to show that it is able to adaptively derive assistive torques even for multiple-joint motion.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.