Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological “fusion pores” can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.     

Surface acidic sites on supported titanium-magnesium catalyst for propylene polymerization: ESR study via adsorption of stable nitroxyl radicals

ESR was used for studying the interaction between the stable nitroxyl radical TEMPO (2,2,6,6-tetramethylpyperidin-1-oxyl) and the titanium-magnesium catalysts TiCl₄/MgCl₂ and TiCl₄/MgCl₂ · nD (D is either diisobutyl phthalate or 2,2-diisobutyl-1,3-dimethoxypropane), as well as TiCl₄/MgCl₂ · nD treated with triethylaluminium. In all cases, only part of the surface titanium complexes (18–53 mol-%) exhibit Lewis acid properties and can interact with TEMPO. The portion of titanium complexes with acidic properties depends on the composition and preparation of the catalysts, and it decreases upon treatment with triethylaluminium.     

Sequence-based typing of ade B as a potential tool to identify intraspecific groups among clinical strains of multidrug-resistant Acinetobacter baumannii

Sequence analysis of an 850-bp fragment internal to the aspecific drug efflux gene ade B revealed 11 sequence types (STs) among a collection of 50 multidrug-resistant Acinetobacter baumannii (MDRAB) strains, including members of pan-European clones I, II, and III. The delineation of STs conformed with the intraspecific grouping of these strains previously determined by different DNA fingerprinting methods. Larger strain collections need to be screened to further explore the potential of sequence-based ade B typing as a universal tool for the monitoring of MDRAB clones.     

Blogging Your PACS

Acquiring, implementing, and maintaining a picture archiving and communication system (PACS) is an enduring and complex endeavor. A large-scale project such as this requires efficient and effective communication among a large number of stakeholders, sharing of complex documentation, recording ideas, experiences, and events such as meetings, and project milestones to succeed. Often, mass-market technologies designed for other purposes can be used to solve specific complex problems in healthcare. In this case, we wanted to explore the role of popular weblogging or “blogging” software to meet our needs. We reviewed a number of well-known blog software packages and evaluated them based on a set of criteria. We looked at simplicity of installation, configuration, and management. We also wanted an intuitive, Web-based interface for end-users, low cost of ownership, use of open source software, and a secure forum for all PACS team members. We chose and implemented the Invision Power Board for two purposes: local PACS administrative purposes and for a national PACS users' group discussion. We conclude that off the shelf, state-of-the-art, mass-market software such as that used for the currently very popular purpose of weblogging or “blogging” can be very useful in managing the variety of communications necessary for the successful implementation of PACS.     

Development of the NADPH-diaphorase-positive neurons in the sympathetic ganglia

Changes in the distribution of NADPH-diaphorase (NADPH-d) were studied in neurons of the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglia (CG) in newborn, 10-, 20-day-old, 1-month-old, 2-month-old and 6-month-old rats, mice and kittens. NADPH-d-positive neurons were revealed in all sympathetic ganglia in kittens but not in rodents from birth onwards. In kittens, the largest population of NADPH-d-positive cells was found in the SG, the smallest in the SCG (<1%) and we observed only a few cells in the CG. The proportion of NADPH-d-positive cells in the SG increased from 3.1±0.15% in newborn kittens to 9.3±0.63% in 20-day-old animals and decreased further from 8.1±0.75% in 30-day-old kittens to 3.4±0.54% in 2-month-old animals. The content of NADPH-d-positive cells in the CG and SCG did not change during development. There were no differences in cross-sectional area between neurons located in different ganglia of animals from the same age group under study. We conclude that the development of NADPH-d-positive neurons in different sympathetic ganglia has its own time dynamics and is completed by the end of the second month of life.     

Distribution of tetracycline resistance genes in genotypically related and unrelated multiresistant Acinetobacter baumannii strains from different European hospitals

The presence of tetracycline (TET) resistance genes was investigated in 49 genotypically related and unrelated multidrug-resistant Acinetobacter baumannii (MDRAB) strains from European hospitals including representatives of pan-European clones I and II. Except for one strain, all MDRAB strains displayed resistance to tetracycline (MIC range of 16 to > 512 microg/ml) but were susceptible (MIC < 4 microg/ml) or exhibited intermediate resistance (MIC of 4-8 microg/ml) to minocycline (MIN). In 37 strains, either tet(A) or tet(B) was detected and one of these strains possessed both tet(A) and tet(M). In addition, all MDRAB strains contained the aspecific efflux gene adeB irrespectively of whether they harbored tet genes or not. Repetitive DNA element (rep)-PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] revealed that strains previously assigned to pan-European clones I and II were grouped into two separate clusters. In addition, these clusters also contained strains that had not been typed previously, indicating that (GTG)5-PCR is a valuable method for recognizing putative new members of MDRAB clones. Most, but not all, members of clones I and II were linked to the presence of either tet(A) or tet(B) and displayed different levels of TET resistance with MIC values of 32 to > 512 microg/ml and > 512 microg/ml, respectively. Of these two genes only tet(B) encodes an efflux of both TET and MIN, which was reflected by the relatively high MIC values for MIN (4 microg/ml) shown by the majority of the tet(B)-carrying clone II strains as opposed to the low MIC values for MIN (< 1 microg/ml) displayed by most tet(A)-containing clone I strains. Collectively, our phenotypic and genotypic resistance data support the therapeutic evaluation of second-generation tetracyclines like MIN as promising agents for treating MDRAB infections.     

Extracellular ionic and volume changes: The role in glia—Neuron interaction

Activity-related changes in extracellular K⁺ concentration ([K⁺]_e), pH (pH_e) and extracellular volume were studied by means ofion-selective microelectrodes in the adult rat spinal cord in vivo and in neonatal rat spinal cords isolated from pups 3–14 days of age (P3–P14). Concomitantly with the ionic changes, the extracellular space (ECS) volume fraction (α), ECS tortuosity (λ) and non-specific uptake (k′), three parameters affecting the diffusion of substances in nervous tissue, were studied in the rat spinal cord gray matter. In adult rats, repetitive electrical nerve stimulation (10–100 Hz) elicited increases in [K⁺]_e of about 2.0–3.5 mm, followed by a post-stimulation K⁺-undershoot and triphasic alkaline-acid-alkaline changes in pH_e with a dominating acid shift. The ECS volume in the adult rat occupies about 20% of the tissue, α = 0.20 ± 0.003, λ = 1.62 ± 0.02 and k′ = 4.6 ± 0.4 × 10⁻³s⁻¹ (n = 39). In contrast, in pups at P3–P6, the [K⁺]_e increased by as much as 6.5 mm at a stimulation frequency of 10 Hz, i.e. K⁺ ceiling level was elevated, and there was a dominating alkaline shift. An increase in [K⁺]_e as large as 1.3–2.5 mm accompanied by an alkaline shift was evoked by a single electrical stimulus. The K⁺ ceiling level and alkaline shifts decreased with age, while an acid shift, which was preceded by a small initial alkaline shift, appeared in the second postnatal week. In pups at P1–P2, the spinal cord was X-irradiated to block gliogenesis. The typical decrease in [K⁺]_e ceiling level and the development of the acid shift in pH_e at P10–P14 were blocked by X-irradiation. Concomitantly, continuous development of glial fibrillary acidic protein positive reaction was disrupted and densely stained astrocytes in gray matter at P10–P14 revealed astrogliosis.The alkaline, but not the acid, shift was blocked by Mg²⁺ and picrotoxin (10⁻⁶m). Acetazolamide enhanced the alkaline but blocked the acid shift. Furthermore, the acid shift was blocked, and the alkaline shift enhanced, by Ba²⁺, amiloride and SITS. Application of glutamate or gamma-aminobutyric acid evoked an alkaline shift in the pH_e baseline at P3–P14 as well as after X-irradiation. The results suggest that the activity-related acid shifts in pH_e are related to membrane transport processes in mature glia, while the alkaline shifts have a postsynaptic origin and are due to activation of ligand-gated ion channels.At P4–P6, the ECS volume was almost double that in adult rats, α = 0.37 ± 0.01 (n = 17), the ECS tortuosity was significantly higher, λ = 1.78 ± 0.02, while the non-specific uptake was not significantly different, k′ = 3.61 ± 0.56 × 10⁻³ s⁻¹. The α gradually decreased to about 24% at P12. In adult rats, electrical or adequate stimulation evoked a shrinkage of the extracellular space by 20–50%, while no significant changes in ECS volume were found in P3–P6. We conclude that the [K⁺]_e ceiling level, character of the pH_e transients, the size of the ECS volume and the activity-related ECS shrinkage are closely related to gliogenesis.     

Structural study of compounds modelling elementary polymer units, 6. X-ray structural and quantum-chemical investigation of the cis- and trans-isomeric models of the polynaphthoylenebenzimidazole elementary units

The X-ray structural investigation of the naphthoylenebis-(benzimidazole) ( 1 ) cis- and trans-isomers as models for the cis and trans elementary units of the corresponding poly(naphthoylene-benzimidazoles) (PNBI) was carried out. The correctness of the previous identification of the isomers was confirmed, and exact geometrical parameters of the PNBI elementary units (required, in particular, for the calculation of the Kuhn segment of PNBI) were determined. Quantum-chemical semi-empirical calculations of the ground and excited electronic states of the isomers were carried out. Both experimental and theoretical data confirm a larger stability of the planar π-conjugated structure of the trans-isomer in comparison with the cis-isomer in the ground as well as in the excited electronic state. Crystals of the cis- and trans-isomers obtained from trifluoroacetic acid solutions are the solvates of bis(trifluoroacetates) of diprotonated 1 with an extremely high content of the solvating trifluoroacetic acid. This peculiarity is favourable for modelling the main characteristics of the interaction between PNBI and protonic polar solvent molecules, viz. preferred types of H-bonding and a possibility of interaction of the electronic lone pairs of the solvent molecules with the π-electronic systems of the elementary units of PNBI by charge transfer complexes.