X-ray microcomputed tomography as a tool for the investigation of the biodistribution of magnetic nanoparticles.

Computed tomography is a widely used technique to study the inner structure of opaque samples using the material-dependent attenuation of x-rays. Microcomputed tomography follows the same principles used for conventional medical CT scanners, but improves the spatial resolution to a few micrometers. As an example for the application of x-ray microtomography, the study of the 3D biodistribution of magnetic nanoparticles in tumoral tissue after minimal invasive cancer therapy, which is one of the crucial factors for this kind of therapy, is presented in this article. In particular, the possibilities and problems resulting from the use of different sources of radiation--synchrotron and x-ray tubes, respectively--will be discussed.

Microcomputed tomography analysis of ferrofluids used for cancer treatment.

In order to reduce the side effects generated by the most common cancer treatment therapies, chemo- and radiotherapy, two new approaches are being investigated. These new approaches are magnetic drug targeting (MDT) and magnetic hyperthermia, and are based on the use of magnetic nanoparticles. In the first one, these magnetic nanoparticles are used as drug carriers and the success of the treatment depends on the correct distribution of the drug within the tumour tissue. Computed tomography analysis has been performed on tumour tissue after MDT in order to find out the distribution of the nanoparticles. The measurements have been carried out in two different laboratories, one based on a synchrotron beamline and another one with a cone x-ray source. First results show that the drug carriers form clusters within the tumour tissue.

Time-resolved confocal fluorescence imaging and spectrocopy system with single molecule sensitivity and sub-micrometer resolution.

We present novel technical features and results from a two channel confocal fluorescence lifetime microscope, which allows to efficiently investigate fluorescence dynamics down to the single molecule level. The MicroTime 200 time-resolved fluorescence microscope offers a multicolor excitation where different picosecond diode lasers are used. For imaging and positioning purposes we utilize a compact Piezo scanner which allows, due to a novel scanning algorithm and synchronisation technique, a superior movement and positioning accuracy. The data acquisition is completely based on time-correlated single photon counting, where every photon is detected and stored individually with its specific timing information (Time-Tagged Time-Resolved mode). This multiparameter data acquisition scheme offers the opportunity to analyse the parameter dependencies in a multitude of different ways. Standard intensity analysis can be used to reconstruct 2D-images or the temporal evolution (time trace) of the fluorescence of a single spot. The information from the two distinct detector channels additionally allows to investigate the polarisation of the emitted light or its spectral composition, for example for analysis of Fluorescence Resonance Energy Transfer (FRET). The timing information down to a picosecond scale offers the possibility not only to reconstruct fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function of any single spot of interest. The flexible multichannel detector scheme enables in this case also a cross-correlation between spectrally separated parts of the emission light, or even identical parts of the fluorescence to eliminate detector artifacts. The photon arrival coincidence analysis can also be expanded in the sub-ns range to study fluorescence antibunching in the fluorescence emission of single molecules. The ability of combining these different pieces of temporal information allows the construction of extremely powerful analysis methods and assays. We demonstrate a variety of these capabilities with results obtained from fluorescently labeled latex beads, biological samples, and single molecules excited in the blue or red wavelength region.

Dynamics of DNA replication factories in living cells.

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

Functional links between nuclear structure, gene expression, DNA replication, and methylation.

Over the last decades it became clear that mammalian nuclei are highly organized. Nuclear processes like DNA replication and RNA metabolism take place in distinct subnuclear foci, which are enriched for enzymes involved in the corresponding biochemical reactions. This colocalization of functions with their respective factors is often referred to as functional organization of the nucleus. This organization is achieved by assembly of different enzymes and regulatory factors into high-molecular-weight complexes that are tethered to insoluble nuclear structures. Recently, several links between nuclear structure, gene expression, DNA replication, and methylation have been described that illustrate the interrelation of higher-order structures and nuclear functions. New insights into the functional organization of the nucleus and how it could explain the high precision and overall coordination of nuclear processes are discussed.

Intranuclear targeting of DNA replication factors.

Mammalian nuclei are highly organized into functional compartments. Major nuclear processes like DNA replication and RNA processing take place in distinct foci. These microscopically visible foci are formed by the assembly of, for example, DNA replication factors and associated proteins into megadalton complexes often referred to as protein machines or factories. Thus far, two proteins, DNA ligase I and DNA methyltransferase (DNA MTase), have been analyzed in greater detail. In both cases, the assembly process appears to be controlled by distinct targeting sequences that were attached to the catalytic protein core in the course of evolution and mediate the association with replication factories in mammalian cells. The dynamics of these nuclear structures throughout the cell cycle are analyzed using green fluorescent protein (GFP). Further studies are needed to elucidate the architecture, regulation, and role of these subnuclear structures.

Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

Biotechnological aspects of the production of human pro-kallikrein using the AcNPV-baculovirus-expression system.

We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.

Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells.

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.

Expression of human salivary-gland kallikrein in insect cells by a baculovirus vector.

A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.

[The crystallization behavior of chromatographically pure swine insulin]. / Untersuchungen zum Kristallisationsverhalten von chromatographisch gereinigtem Schweineinsulin.

Crystallization tests are performed with porcine insulin previously purified by single chromatography to produce Lente insulins. The following parameters are changed under the test: crystallization temperature, stirring rate, pH of crystallization, and the period from the beginning of insulin precipitation until the isoelectric point is reached. The effects of these parameters on crystallization kinetics, crystal size and crystal size distribution are being studied.

[Quantitative determination of crystallin insulin in insulin-zinc suspensions as an in-process control]. / Quantitative Bestimmung des kristallinen Insulins in Insulin-Zink-Suspensionen als Inprozess-Kontrolle.

A method is described for the quantitative determination of amorphous and crystalline insulin contents in insulin zinc suspensions. The different resolving rates of the modifications in acid dilutions are utilized for the test. Measurements are made by UV spectroscopy. Information is given on the application of this method for the analysis of the course of insulin crystallization and it is compared with the USP XXI method.

The acoustocardiogram: a noninvasive method for measuring heart rate of avian embryos in ovo.

A method is presented for measuring the heart rate of avian eggs noninvasively during the last half of incubation. The technique involves briefly placing an egg in tightly sealed vessel containing an inexpensive condenser microphone. The amplified output of the microphone, termed the acoustocardiogram (ACG), is nearly sinusoidal in shape and synchronous with the electrocardiogram. The ACG can also be obtained by mounting the microphone directly on the shell with Plasticine. The method offers advantages over previously described techniques in simplicity, low cost, and noninvasiveness.

[The effect of incorporation technics of water soluble macromolecular binders on the properties of fluidized-bed dried granules]. / Zum einfluss der Einarbeitungstechnik von wasserlöslichen makromolekularen Bindemitteln auf die Eigenschaften von Wirbelschichtgranulaten und der daraus hergestellten Komprimate.

Five binders characterized by particle size and dissolution rate have been studied in order to evaluate their qualification for fluidized-bed granulation. For this purpose they were applied as powder and solution, respectively. Quality parameters of the granules and tablets obtained were compared. Compared with solutions of binders the dry incorporation of the binder led to a slower growth of the granules mainly depending on the dissolution rate of the binders. Due to this fact the formulations showed a decreased strength except PVP (high dissolution rate) which causes an increased growth of the granules at the same time. A size reduction of binders with a low dissolution rate led to tablets with characteristics of strength similar to the findings when solutions of binders were used, whereas the growth of granules remained uninfluenced.

Diffusive resistance of avian eggshell pores.

Resistance to gas diffusion through the avian eggshell resides in the microscopic pores which penetrate the shell. We calculated the resistance to water vapor diffusion of individual pores in the shells of 23 species of avian eggs, based on measurements of pore dimensions taken from drawings of 321 pore casts published by Tyler (1962, 1964, 1965) and Tyler and Simkiss (1959). Diffusive resistances were calculated from Fick's first law, using a 100-segment model of each pore. In addition, we added 2 series resistances, calculated from Stefan's law, to account for boundary layer resistances at the inner and outer pore apertures. Convective resistances for the same 100-segment model were computed from Poiseuille's law. A special, symmetrically branching model is presented for the diffusive resistance of the branched pores of ostrich eggshells, based on the drawings of Tyler and Simkiss (1959). The total aperture resistance was less than 6.2% of total pore resistance, while the outside aperture effect was on average only 1.5%. The calculated average pore conductance for all species was 5.4 micrograms (day Torr)-1, about three times higher than the average value of 1.6 micrograms (day Torr)-1 obtained by dividing measured shell conductance by the number of pores (Ar and Rahn, 1985). A possible explanation for this discrepancy is advanced. However, it is to be noted that in spite of the discrepancy, both calculated and functional values of pore conductance appear to be independent of egg mass.

Air cell gas tensions of the chick embryo at sea level and altitude: the contribution of Aggazzotti, 1914.

This historical note draws attention to the forgotten but remarkable study of A. Aggazzotti of Italy who in 1914 described for the first time the changes in O2 and CO2 tensions during the course of development of the chick embryo and repeated these observations at an altitude of 3000 m. When his gas tension data are recalculated and compared with those obtained by Wangensteen et al. at sea level and altitude, 60 years later, the agreement is good.

A blood-gas nomogram of the chick fetus: blood flow distribution between the chorioallantois and fetus.

This paper presents equations for quantifying the relationships between the O2 and CO2 concentrations and tensions in the blood of the 18-day chick fetus. A blood-gas nomogram showing these relationships is presented. Starting with the reported chorioallantoic artery and vein gas tensions and using the blood-gas equations, the range of embryonic arterial and venous gas tensions as well as the distribution of the cardiac output and the degree of mixing between the chorioallantoic and embryonic circulations are explored. It is concluded that at least 65% of the blood in the chorioallantoic artery consists of blood of embryonic mixed venous composition. A model of the blood flow distribution is proposed in which chorioallantoic and embryonic flows are equal, with 70% of the blood returning from the tissues of the embryo going to the chorioallantois and vice versa.

Regional differences in diffusive conductance/perfusion ratio in the shell of the hen's egg.

Are both gas exchange and gas tensions uniform in different regions of the developing hen's egg? To answer this question we measured the O2 uptake and CO2 production of the whole egg, and at the same time the O2 and CO2 tensions of the air cell. The gas exchange ratio (R) of the whole egg differed from R calculated from air cell PO2 and PCO2 values, in agreement with the findings of Visschedijk [Br. Poultry Sci. 9:173-184 (1968)], who measured gas exchange separately over both the air cell region and the remainder of the egg. We constructed a diffusive shell conductance/perfusion (G/Q) line on the O2-CO2 diagram from a blood nomogram for the chick embryo in late development [Olszowka et al., Fed. Proc. 46:512 (1987)], and used this to analyze our results. The G/Q ratio for the area of shell over the air cell differs from that for the remainder of the egg. Our analysis permits us to calculate, for each area, the regional shell conductance, blood flow, and O2 and CO2 tensions in the gas spaces between the shell and the chorioallantoic capillaries.

Regional differences in shell conductance and pore density of avian eggs.

Minidesiccators were attached to the middle and to the blunt and pointed ends of avian eggs of six different species and from their mass change over time the regional shell gas conductance was determined. Later the pore density and shell thickness of these areas were measured. All species showed a decline in regional shell conductance and pore density from the blunt end to the pointed end. With the blunt end over the air cell as a reference point, the regional conductance of the middle and the pointed end declined to 88 and 63%, while pore density fell to 81 and 63%, respectively. The six species represent four orders of birds, and the results suggest that differences in regional conductance may be a relatively common characteristic of bird eggs, that differences in regional pore conductance are proportional to the pore density, and that therefore the conductance of individual pores in any one species is relatively constant.