Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.     

The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel

C5b-9(m) complexes were incorporated into lecithin liposomes and subjected to proteolysis in the presence of DTT to remove the externally oriented annulus. Liposomes were recovered that selectively carried the membrane-bound, thin-walled cylindrical portion of the C5b-9(m) complex. The presence of DTT during proteolysis enhanced peptide bond cleavage in the C5b-9(m) complex. All C5-C9 components were degraded to lower m.w. fragments. A protease-resistant, but hydrophilic 85 to 86,000-dalton polypeptide derivative of C5, possibly representing the C5 beta-chain, was recovered in the fluid phase. This component is not intimately associated with the target lipid bilayer. Immunochemical analyses yielded evidence for the existence of minor C5-C9 antigenic determinants on the membrane-bound C5b-9(m) residue. SDS polyacrylamide gel electrophoreses of liposomes carrying the C5b-9(m) residues revealed the persistence of at least six major polypeptides of approximately m.w. 50,000, 45,000, 40,000, 38,000, 20,000, and 16,000. The data are interpreted to indicate that multiple protease-resistant polypeptide chains derived from several terminal C components participate in formation of the trans-membrane C channel.     

Representation of DNA sequences by use of helical wheels to identify pattern formation with protein binding potential

Similar to the protein segments with helical potential the three-dimensional structures of DNA sequences can be represented by a two-dimensional projection along the axis of the double helix. We will call this projection according to Schiffer and Edmundson "helical wheel". In the first place the wheels are employed here as graphical restatements of known sequences which represent binding sites of restriction-modification enzymes. Specific recognition of these sites has to be based on sequence related multi contact interactions of weak bonds. Because of the spatial and directional characteristics of H-bonds we will utilize the pattern formation of H-bond donor--and acceptor groups protruding into the major groove of the helix to identify the particular sequences. The characteristics of these patterns can be readily visualized and compared by examination of these wheels.     

Codification and evolution of experimentally observed specific recognition sites for restriction enzymes on DNA

The list of published restriction endonucleases along with their substrates provides an excellent data base for the evaluation of the evolution and codification of the key elements for specific recognition sites on the DNA. In this paper the considerations will be limited to palindromic tetramer-, pentamer-, and hexamer-sequences. It is basically assumed that each base pair within these sequences has to be recognized by directionally unique bidentate hydrogen bonds either within the plane of the base pair or by bridging the appropriate H-bond donor/acceptor groups of the neighbouring bases of the same strand. Thus sequence specificity is mediated by twelve (eight) H-bonds, originating from the protein recognition modules. Besides a pronounced preference for GC base pairs expressed by their high frequency in the most abundant sequences, serving the need of maximal thermodynamic stability of the double helical substrates, it can also be shown that the stacking of consecutive bases within the recognition site sequences plays a major role in shaping the particular DNA/protein interface. Finally it will be demonstrated that the full set of sequences discussed in this paper can readily be derived by stepwise expanding the vocabulary of three simple tetrameric sequences by inserting single base pairs into the centre of a minimal sequence, thus creating all the published pentameric restriction sites, or by inserting/adding two GC base pairs in a palindromic way, thus creating the known multiplicity of hexameric sites.     

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

An association of early puberty with disordered eating and anxiety in a population of undergraduate women and men

Eating and anxiety disorders are more prevalent in females, increase during adolescence, and are associated with early pubertal development. This study examined whether timing of puberty onset is associated with disordered eating and anxiety in a large sample of postpubertal male and female undergraduate students. Self-report questionnaires assessed timing of puberty, disordered eating, anxiety, alcohol use, personality, and sensation seeking. Females scored significantly higher on measures of disordered eating (binge eating, dietary restraint, eating concerns, and weight and shape concerns) and anxiety (state and trait anxiety) than did males. In addition, early maturing women and men scored significantly higher on measures of disordered eating and anxiety than on time or late maturing women and men. Measures of alcohol use, sensation seeking, and personality characteristics differed in males and females but did not vary with pubertal timing. Findings suggest that early puberty is associated with disordered eating and anxiety, and this association may be due to an organizational effect of pubertal hormones. Despite important differences in body fat composition, both males and females experiencing early puberty had an increased incidence of disordered eating. The fact that early puberty was associated with increased eating and anxiety symptoms in both sexes suggests that puberty may influence these symptoms through both biological and psychosocial mechanisms.     

Context-dependent ‘safekeeping’ of foraging tools in New Caledonian crows

Several animal species use tools for foraging, such as sticks to extract embedded arthropods and honey, or stones to crack open nuts and eggs. While providing access to nutritious foods, these behaviours may incur significant costs, such as the time and energy spent searching for, manufacturing and transporting tools. These costs can be reduced by re-using tools, keeping them safe when not needed. We experimentally investigated what New Caledonian crows do with their tools between successive prey extractions, and whether they express tool ‘safekeeping’ behaviours more often when the costs (foraging at height), or likelihood (handling of demanding prey), of tool loss are high. Birds generally took care of their tools (84% of 176 prey extractions, nine subjects), either trapping them underfoot (74%) or storing them in holes (26%)—behaviours we also observed in the wild (19 cases, four subjects). Moreover, tool-handling behaviour was context-dependent, with subjects: keeping their tools safe significantly more often when foraging at height; and storing tools significantly more often in holes when extracting more demanding prey (under these conditions, foot-trapping proved challenging). In arboreal environments, safekeeping can prevent costly tool losses, removing a potentially important constraint on the evolution of habitual and complex tool behaviour.     

MicroRNAs in systemic rheumatic diseases

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.