Projection maps of three members of the KdgM outer membrane protein family

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7 Å. All three proteins exhibit a similar putative β-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using β-barrel specific prediction algorithms. The predicted transmembrane β-barrels have a high similarity in the arrangement of the putative β-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

Structural models of the supramolecular organization of AQP0 and connexons in junctional microdomains

Membrane proteins perform many essential cellular functions. Over the last years, substantial advances have been made in our understanding of the structure and function of isolated membrane proteins. However, like soluble proteins, many membrane proteins assemble into supramolecular complexes that perform specific functions in specialized membrane domains. Since supramolecular complexes of membrane proteins are difficult to study by conventional approaches, little is known about their composition, organization and assembly. The high signal-to-noise ratio of the images that can be obtained with an atomic force microscope (AFM) makes this instrument a powerful tool to image membrane protein complexes within native membranes. Recently, we have reported high-resolution topographs of junctional microdomains in native eye lens membranes containing two-dimensional (2D) arrays of aquaporin-0 (AQP0) surrounded by connexons. While both proteins are involved in cell adhesion, AQP0 is a specific water channel whereas connexons form cell–cell communication channels with broad substrate specificity. Here, we have performed a detailed analysis of the supramolecular organization of AQP0 tetramers and connexon hexamers in junctional microdomains in the native lens membrane. We present first structural models of these junctional microdomains, which we generated by docking atomic models of AQP0 and connexons into the AFM topographs. The AQP0 2D arrays in the native membrane show the same molecular packing of tetramers seen in highly ordered double-layered 2D crystals obtained through reconstitution of purified AQP0. In contrast, the connexons that surround the AQP0 arrays are only loosely packed. Based on our AFM observations, we propose a mechanism that may explain the supramolecular organization of AQP0 and connexons in junctional domains in native lens membranes.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

Effects of three benzimidazoles on growth, general morphology and ultrastructure of Tritrichomonas foetus

Tritrichomonas foetus is a venereal pathogen of cattle, which causes infertility, early embryonic death or abortion. In order to evaluate the potential trichomonicidal activity of benzimidazoles, the effects of thiabendazole, mebendazole and albendazole were analyzed on the multiplication, general morphology and ultrastructure of T. foetus. It was found that mebendazole presented the highest IC(50%) (2.3 microM), when compared with albendazole (IC(50%)=9.4 microM) and thiabendazole (IC(50%)=142.6 microM), and that such effects were irreversible. Concerning microscopic analysis, thiabendazole- and mebendazole-treated cells presented increased volume, internalization of the flagella, disruption or multiplication of the nucleus, multiple organelles and cytoplasmic vacuolization. Albendazole-treated cells exhibited slight alterations, because the parasite became slightly rounded, its flagella were not internalized but the cytoplasm was vacuolated. Mebendazole was indeed highly effective as an in vitro trichomonicidal agent, and this might open up new possibilities for the use of mebendazole in the therapy of bovine trichomoniasis.     

Enhanced efficiency due to the use of achiral additives in the optical resolution of 1-phenylethylamine by its glutaric acid derivative

Racemic 1-phenylethylamine was optically resolved by its own derivative formed with glutaric acid namely (+)-(R)-N-(1-phenylethyl)glutaramic acid. The amide acid resolving agent was synthesized from (+)-(R)-1-phenylethylamine by N-derivatization. The glutaric acid derivative was the next in a homologous series of dicarboxilic acid derivatized resolving agents of racemic 1-phenylethylamine. Resolution results obtained with the oxalic, malonic, and succinic acid derivatives were previously discussed(1). Each of the above derivative resolving agents could be successfully applied as resolving agents of 1-phenylethylamine. The efficiency of the present optical resolution using (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent was remarkably inferior to the results obtained by its shorter chained homologues(1). Use of achiral additives, like urea, thiourea, N-methylurea, and N,N'-dimethylurea caused large increase in the efficiency of the resolution by (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent. Precipitated salts obtained in the resolutions performed in the presence of the additives were investigated by thermoanalysis, X-ray powder diffraction, and optical microscopy. Based on the analytical data, the improvement of the resolution results was attributed to the influence of the additives on the crystal nucleation processes of the diasteromeric salts.     

Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: An ultrastructural study

A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain.     

Phloxine B, a versatile bacterial stain

The data presented suggest that Phloxine B, a color additive for food, drugs, and cosmetics has a potential use as a nontoxic, faster (<2 min), inexpensive (350 tests for <1 cent material) and simpler to use alternative to Gram staining. Using Phloxine B staining it was possible to differentiate among gram-negative and gram-positive bacteria by visual determination under normal room lighting, light microscopy, fluorescence microscopy and confocal microscopy. This work demonstrated that Phloxine B can be used as a differential versatile bacterial stain and establishes a correlation between the staining properties of the dye and its bactericidal effect.