Antiestrogenic and anticancer activities of peptides derived from the active site of alpha‐fetoprotein

Cyclo[EKTOVNOGN] (AFPep), a cyclic 9‐amino acid peptide derived from the active site of alpha‐fetoprotein, has been shown to prevent carcinogen‐induced mammary cancer in rats and inhibit the growth of ER⁺ human breast cancer xenografts in mice. Recently, studies using replica exchange molecular dynamics predicted that the TOVN region of AFPep might form a dynamically stable putative Type I beta‐turn, and thus be biologically active without additional amino acids. The studies presented in this paper were performed to determine whether TOVN and other small analogs of AFPep would inhibit estrogen‐stimulated cancer growth and exhibit a broad effective‐dose range. These peptides contained nine or fewer amino acids, and were designed to bracket or include the putative pharmacophoric region (TOVN) of AFPep. Biological activities of these peptides were evaluated using an immature mouse uterine growth inhibition assay, a T47D breast cancer cell proliferation assay, and an MCF‐7 breast cancer xenograft assay. TOVN had very weak antiestrogenic activity in comparison to AFPep's activity, whereas TOVNO had antiestrogenic and anticancer activities similar to AFPep. OVNO, which does not form a putative Type I beta‐turn, had virtually no antiestrogenic and anticancer activities. A putative proteolytic cleavage product of AFPep, TOVNOGNEK, significantly inhibited E₂‐stimulated growth in vivo and in vitro over a wider dose range than AFPep or TOVNO. We conclude that TOVNO has anticancer potential, that TOVNOGNEK is as effective as AFPep in suppressing growth of human breast cancer cells, and that it does so over a broader effective‐dose range. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 μM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.     

SOD1 Mutations Targeting Surface Hydrogen Bonds Promote Amyotrophic Lateral Sclerosis without Reducing Apo-state Stability

In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.     

Do kinematic models reduce the effects of soft tissue artefacts in skin marker-based motion analysis? An in vivo study of knee kinematics

We investigated the effects of including kinematic constraints in the analysis of knee kinematics from skin markers and compared the result to simultaneously recorded trajectories of bone pin markers during gait of six healthy subjects. The constraint equations that were considered for the knee were spherical and revolute joints, which have been frequently used in musculoskeletal modelling. In the models, the joint centres and joint axes of rotations were optimised from the skin marker trajectories over the trial. It was found that the introduction of kinematic constraints did not reduce the error associated with soft tissue artefacts. The inclusion of a revolute joint constraint showed a statistically significant increase in the mean flexion/extension joint angle error and no statistically significant change for the two other mean joint angle errors. The inclusion of a spherical joint showed a statistically significant increase in the mean flexion/extension and abduction/adduction errors. In addition, when a spherical joint was included, a statistically significant increase in the sum of squared differences between measured marker trajectories and the trajectories of the pin markers in the models was seen. From this, it was concluded that both more advanced knee models as well as models of soft tissue artefacts should be developed before accurate knee kinematics can be calculated from skin markers.     

Two dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.     

Production of ethanol from mannitol by Zymobacter palmae

Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic mannitol medium with a yield of 0.38 g ethanol (g mannitol)⁻¹. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57. Received 27 June 1999/ Accepted in revised form 23 September 1999     

Reconstitution of barley photosystem I reveals that the N-terminus of the PSI-D subunit is essential for tight binding of PSI-C

Removal of the peripheral subunits PSI-C, -D and -E from the photosystem I (PSI) complex of barley requires a urea treatment much harsher than required to remove the similar subunits from cyanobacterial PSI. The resulting PSI barley core was reconstituted by addition of the E. coli expressed subunits PSI-C and -D, and PSI-E isolated from barley. Western blotting, flash photolysis and NADP⁺ photoreduction measurements demonstrated complete and specific removal of the three subunits from the core and efficient reconstitution of the complex after addition of PSI-C, -D and -E. Flash photolysis reveals that PSI-D is essential for binding of functional PSI-C to the PSI core. An N-terminally truncated barley PSI-D lacking 24 amino acid residues and thus being without the N-terminal extension characteristic for higher plant PSI-D proteins reconstitutes the PSI core to 50% of the level obtained with intact PSI-D as demonstrated by flash photolysis and NADP⁺ photoreduction measurements. Cyanobacterial PSI-D is functionally equivalent to truncated barley PSI-D with respect to its activity to reconstitute the PSI core. This shows that the N-terminal extension of plant PSI-D plays a key role in binding PSI-C to the core. The plant-specific N-terminus of PSI-D is hypothesized to execute its function through interaction with a plant-specific PSI subunit, possibly PSI-H. An anchoring function of the N-terminus of PSI-D would also explain the harsh treatment needed to obtain a plant PSI core. PSI-E is important for efficient NADP⁺ reduction but does not influence electron transfer to iron-sulphur centres A/B nor binding of PSI-C. The enhancing effect of PSI-E on NADP⁺ reduction is independent of the presence of the N-terminus of PSI-D.     

MOLECULAR SYSTEMATICS OF THE XANTHOPHYCEAE

The Corallinales includes ca. 40 genera of calcified red seaweeds. Species are of two distinct morphotypes; those that possess genicula (uncalcified nodes) and those that lack genicula. Most nongeniculate species take the form of crusts. The presence (or absence) of genicula, secondary pit connections, and tetrasporangial conceptacle features have traditionally been used as key characters for delimiting coralline subfamilies. In this study, nuclear encoded 18S and 26S r RNA gene sequences were determined and used to reexamine relationships among coralline taxa. Separate and combined phylogenetic analyses of these data yielded similar trees in which four major lineages are resolved. Heydrichia and Sporolithon (Sporolithaceae) are positioned at the base of the tree and appear to be distantly related to other species examined. Within the Corallinaceae, the nongeniculate Melobesioideae is resolved as a monophyletic group. All members of this subfamily produce mutiporate tetraspoangial conceptacles. The Corallinoideae, which are characterized by unizonate genicula, are resolved as sister to a clade containing species placed in the Lithophylloideae, Mastophoroideae and Metagoniolithoideae. The molecular data indicate that geniculate and nongeniculate species characterized by the presence of secondary pit connections are closely related. For example, both data sets robustly support a sister taxon relationship between Amphiroa and Titanoderma. Our results indicate that: 1) all taxa in which secondary pit connections are present should be referred to the Lithophylloideae and, 2) genicula are nonhomologous structures that are independently derived in Amphiroa, Lithothrix, Metagoniolithon and the last common ancestor of the Corallinoideae.