The Neanderthal taxonomic position: models of intra- and inter-specific craniofacial variation

The Neanderthal taxonomic position is a matter of wide disagreement among paleoanthropologists. Some workers consider this fossil human group to represent a different species, Homo neanderthalensis, while others see it as a subspecies of Homo sapiens. This study developed two models of morphological variation to be applied to a comparison between Neanderthals and modern humans: modern human populations provided a measure of intra-specific variation, while the species and subspecies of Pan provided measures of both intra- and inter-specific morphological differences. Although such an approach has been advocated strongly, it has not been systematically undertaken until recently. The techniques of geometric morphometrics were used to collect data in the form of three-dimensional coordinates of craniofacial landmarks. The data were processed using generalized procrustes analysis, and analyzed by an array of multivariate statistical methods, including principal components analysis, canonical variates analysis and Mahalanobis D(2). The morphological distances between Neanderthals and modern humans, and between Neanderthals and Late Paleolithic/early anatomically modern specimens, are consistently greater than the distances among recent human populations, and greater than the distances between the two chimpanzee species. Furthermore, no strong morphological similarities were found between Neanderthals and Late Paleolithic Europeans. This study does not find evidence for Neanderthal contribution to the evolution of modern Europeans. Results are consistent with the recognition of Neanderthals as a distinct species.     

Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores

The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat. GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat. Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase. GerQ is the only substrate for this transglutaminase identified to date. In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis. These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.     

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (?183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.     

Structure of human alpha1-acid glycoprotein and its high-affinity binding site

Secondary and tertiary structures of human blood alpha(1)-acid glycoprotein, a member of the lipocalin family, have been studied for the first time by infrared and Raman spectroscopies. Vibrational spectroscopy confirmed details of the secondary structure and the structure content predicted by homology modeling of the protein moiety, i.e., 15% alpha-helices, 41% beta-sheets, 12% beta-turns, 8% bands, and 24% unordered structure at pH 7.4. Our model shows that the protein folds as a highly symmetrical all-beta protein dominated by a single eight-stranded antiparallel beta-sheet. Thermal dynamics in the range 20-70 degrees C followed by Raman spectroscopy and analyzed by principle component analysis revealed full reversibility of the protein motion upon heating dominated by decreasing of beta-sheets. Raman difference spectroscopy confirmed the proximity of Trp(122) to progesterone binding.     

Mutually-induced Conformational Switching of RNA and Coat Protein Underpins Efficient Assembly of a Viral Capsid

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T = 3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T = 3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.     

Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the beta-amyloid peptide (Abeta) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Abeta, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Abeta and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Abeta-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Abeta-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.     

Estimation of ABCB1 concentration in plasma membrane

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.     

Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.