Prefixation chromosome banding with heparin

Summary Attempts to achieve chromosomal banding by removal of histone proteins by acid or fixation procedures have failed up to now. Numerous biochemical investigations have shown that heparin has a strong and specific affinity to histone proteins. We describe a procedure by which treatment with heparin of nonfixed and fixed metaphase chromosomes leads to G banding; in the former case the effect is observed after a few minutes of heparin treatment; in the latter, much higher concentrations of heparin and 18–24h incubation are needed. The morphologic effects of heparin treatment, the gradual disruption of chromosomal and nuclear chromatin, are associated with progressive reduction of histone detection (until completely negative) by the alkaline fast green test.     

An enzyme biotechnology for the total utilization of leather wastes

Summary The isolation of protein and fat fractions from a waste of the leather industry is discussed. This represents the main waste of leather production and gives rise to problems of ecological and economical nature. A middle capacity factory deposits about 30 tones of the so called carrion, a source for the production of about 3 t useful products-protein and fat.     

c-Src-dependent activation of the epidermal growth factor receptor and mitogen-activated protein kinase pathway by arsenic. Role in carcinogenesis.

Environmental or occupational exposure to arsenic is associated with a greatly increased risk of skin, urinary bladder, and respiratory tract cancers in arseniasis-endemic areas throughout the world. Arsenic shares many properties of tumor promoters by affecting specific cell signal transduction pathways responsible for cell proliferation. The activation of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated protein kinase (ERK) pathway is important in mediating gene expression related to regulation of cellular growth. In the current studies, we demonstrate that arsenic activates EGFR and ERK in a human uroepithelial cell line. The EGFR phosphorylation by arsenic is ligand-independent and does not involve the major autophosphorylation site Tyr(1173). c-Src activity is also induced by arsenic and is a prerequisite for the EGFR and ERK activation. Consistent with these in vitro observations, exposure of mice to arsenic in drinking water, which has been found previously to be associated with AP-1 activation and epithelial proliferation, induces EGFR and ERK activation in the urinary bladder. This response is also accompanied with an increase in c-Src levels interacting with EGFR. These findings represent a potential pathway for mediating arsenic-induced phenotypic changes in the uroepithelium.     

In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.The Arabidopsis (Arabidopsis thaliana) cell division cycle protein CDC48A was previously shown to interact with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) (Rienties et al., 2005) and to coimmunoprecipitate with SERK1 in Arabidopsis cultured cells and seedlings (Karlova et al., 2006). In living cells the CDC48A protein colocalizes with SERK1 at peripheral endoplasmic reticulum (ER)-based membranes and the plasma membrane (PM). Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) showed that CDC48A interacts with SERK1 at the PM (Aker et al., 2006).CDC48A is a member of the family of AAA ATPases (ATPases associated with various cellular activities), shown to have various functions in cell division, membrane fusions, and in proteasome- and ER-associated degradation (ERAD) of proteins (Woodman, 2003). The role of AAA ATPases is to generate mechanical force to disrupt or fuse molecular structures by means of ATP binding and hydrolysis. The AtCDC48A protein was shown to play a role in ERAD and in membrane fusions (Rancour et al., 2002; Müller et al., 2005). AAA proteins are present as stacked hexameric rings that are stabilized by the binding of ATP. They are only reported to be active in hexameric form. This study aims to determine the predominant form of the fluorescently tagged CDC48A protein in Arabidopsis protoplasts at the peripheral ER and the PM domains where interactions between CDC48A and the SERK1 receptor take place, and, therefore, to determine if SERK1 is associated with the active form of CDC48A.CDC48A is 77% identical to the mammalian homolog vasolin-containing protein (VCP) or p97. Recently, conformational and dynamic changes during the ATP hydrolysis cycle of p97 have been studied by cryo-electron microscopy, crystallography, and small-angle x-ray scattering (for review, see Pye et al., 2006). The monomeric protein contains an N-terminal domain, two AAA domains, and a C-terminal domain (Figs. 1 and ​and3).3). The N domain is important for binding of adaptor proteins and substrates. The two AAA domains D1 and D2 are responsible for binding and hydrolysis of ATP. The D1 and D2 domains form stacked hexameric rings above each other that are connected by a linker. The ATP-binding site in the D1 domain and the D1-D2 linker are responsible for hexamerization, while the ATP-binding site in the D2 domain possesses the major ATPase activity (Wang et al., 2003a). The N domain is flexibly attached to the D1 domain, and projects out of the ring, facilitating binding to other proteins. During ATP hydrolysis, large conformational changes are transmitted from the D2 domain via the D1 domain onto the N domains, due to binding, hydrolysis, and release of the nucleotide (DeLaBarre and Brunger, 2003, 2005). In addition, the pores within the D1 and D2 rings narrow and widen in different nucleotide states. This causes the rings to rotate relative to each other (Davies et al., 2005). The C-terminal tail was not ordered within the crystal structure, and it is therefore unknown whether it stays close to the D2 ring or projects from the core structure toward the N domain.Open in a separate windowFigure 1.Expression in Arabidopsis protoplasts of CDC48A, CDC48A^A1A2, CDC48A^Ndel, and CDC48A^N-D1 mutants all tagged to YFP. YFP is replaced by CrFP. In A the various mutants are shown schematically. Lysates of transfected cells were submitted to SDS-PAGE in B, to native PAGE in C, and probed with anti-YFP anti-serum. The expected sizes of the denatured proteins are 125 kD for full-length (FL) CDC48A- and CDC48A^A1A2-YFP, 96 kD for CDC48A^Ndel-YFP, and 77 kD for CDC48A^N-D1-YFP. Nondenatured hexameric CDC48A-YFP is expected to be between 650 and 750 kD depending on the number of YFP tags.Open in a separate windowFigure 3.Models of the expected hexamers of CDC48A after coexpression of two different protomers. A, Monomeric CDC48A. B, CDC48A fused with CrFP and with YFP (both at the C terminus) are combined and depicted as a hexamer. For simplicity only four fluorophores are drawn at adjacent positions at the C terminus, but in fact these can be randomly positioned at each C domain. C, YFP fused to the N terminus and CrFP to the C terminus. D, YFP and CrFP fused to the N terminus. E and F, CDC48A^N-D1 and CDC48A^Ndel, each depicted as a monomer.For Arabidopsis CDC48A the crystal structure is not known, but in vitro-produced proteins form a hexamer while PUX1 (plant UBX domain-containing protein) facilitates CDC48A oligomer disassembly (Rancour et al., 2004). Up to now hexamerization of AAA ATPases was only shown in vitro or in total cell lysates, studies that do not reveal any spatial information on the oligomerization status of proteins in a living cell. Therefore, the protein was fused to the GFP variant monomeric cerulean fluorescent protein (CrFP) or to yellow fluorescent protein (YFP), and the hexamerization of CDC48A proteins in living cells was monitored by FRET-FLIM. Employing this technique, oligomerization of the CDC48A proteins in living cells was shown to be nonuniform. In addition, intersubunit distances in the oligomeric CDC48A protein could be calculated, revealing that the C-terminal domains stayed in close proximity rather than protruding out of the molecule.It was also shown that SERK1 interacts with the N terminus as well as the C terminus of CDC48A (Aker et al., 2006). After performing a trans-phosphorylation reaction with the SERK1 kinase domain, only Ser-41, a Ser residue found to be in the N domain of CDC48A, was phosphorylated.To investigate the diffusion of the CDC48A protein in cells, fluorescence correlation spectroscopy (FCS), in which fluorescence intensity fluctuations caused by diffusion of fluorescent molecules in and out of a femtoliter volume are monitored in time, was employed. These fluctuations give information about diffusion times of proteins through the volume and, hence, about the size of protein complexes.Our results show that the oligomeric form of CDC48A in living cells is primarily hexameric and that the fluorescently tagged CDC48A is still able to form hexamers. The SERK1 receptor interacts with CDC48A at the same locations where oligomerization of the CDC48A protein is shown. We conclude therefore that SERK1 interacts with the hexameric form of CDC48A. Using FCS the presence of CDC48A in larger protein complexes in vivo was predicted.     

A tale of two oxidation states: bacterial colonization of arsenic-rich environments

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

A study of the interaction of glycine and its oligohomopeptides with formaldehyde and acetaldehyde under possible primitive earth conditions

It is established that glycine and glycine oligohomopeptides interact with formaldehyde and acetaldehyde in a homogeneous weak acid medium (pH 3.3–3.7) at mild temperatures (60–80°C) in the absence of inorganic solid substances. Together with the expected serine and threonine, the formation of alanine, glutamic and aspartic acid, norvaline and isoleucine, as well as four non-protein amino acids is also established. It is suggested that the non-protein amino acids are hydroxymethylserine, hydroxymethylthreonine, hydroxymethylaspartic acid and γ-amino-δ-hydroxyvaleric acid. The modes of formation of all protein and non-protein amino acids are discussed. These results strengthen the probability that similar processes may have been one of the pathways for the prebiotic synthesis of amino acids on primitive Earth.     

o-Diphenol oxidase activity of molluscan hemocyanins

Diphenoloxidase activities of two molluscan hemocyanins, isolated from the marine snails Rapana venosa and garden snails Helix vulgaris were studied using o-diphenol and L-Dopa as substrates. The dimers of H. vulgaris Hc show both, diphenol (K(m)=2.86 mM and K(cat)=4.48) and L-Dopa activity due to a more open active sites of the enzyme and better access of the substrates. The K(m) value of molluscan H. vulgaris Hc is very close to those of Helix pomatia and Sepia officinalis Hcs, but several times higher compared to those of Rapana and Octopus Hcs. Also HvH has a very high enzyme activity compared with other molluscan Hcs. Kinetic measurements with native RvH and both structural subunits, RvH1 and RvH2, show that RvH and only one structural subunit, RvH2, exhibited only o-diphenol activity, but no L-Dopa oxidizing activity.