The inherent properties of DNA four-way junctions: comparing the crystal structures of holliday junctions

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.     

Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites

With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain.     

Effects of emotion-oriented care on elderly people with cognitive impairment and behavioral problems in residential homes

This study investigates the effects of emotion-oriented care on the behavior of elderly people with cognitive impairment and behavioral problems. This approach is mainly based on the validation approach, but uses also insights from other approaches like reminiscence and sensory stimulation. Sixteen homes for the aged with structured day care units were randomly allocated to an intervention or control group. 151 Residents with cognitive impairment and behavioral problems were included in the study. The eight intervention homes received a training program with regard to emotion-oriented care. In the eight control homes usual care was continued. Measurements were performed at baseline and after three, six and twelve months of follow-up (assessment by caregivers and relatives). The primary outcome measure was the change in behavior of the residents. The results of multilevel analyses (overall, subgroup and per protocol) showed no statistically significant, nor clinically relevant effects in favor of the intervention group on the behavioral outcome measures. A possible reason for this is that the intervention did not result in significant contrast between the study groups. There is insufficient scientific evidence yet to justify the implementation of emotion-oriented care in residential homes, for residents with moderate to severe cognitive impairment and behavioral problems, on a large scale. Additional studies are needed in which special attention should be given to the implementation process.     

Possibilities of extraction and characterization of ancient plasma proteins in archaeological bones

Due to the mineral matrix bone proteins are capable of surviving during centuries after inhumation, but cross-linking with other bone proteins as well as fragmentation and complex reactions with humic acids and microorganisms lead to considerable alterations in molecular weight and structure of these proteins. Our group concentrates on polymorphic plasma proteins which diffuse out of the capillary system into the bone matrix where they adsorb to the mineralic substrate. So far, only little is known about the degradation and alteration of these proteins in fossil bones. It has to be evaluated whether the aged proteins still contain some of the information which renders them a valuable tool for forensic questions and population genetics in recent populations. Therefore we tried by modification of already existing methods to expand plasma protein identification and subtyping into the new field of aged plasma proteins.     

Two spatial memories for honeybee navigation

Insect navigation is thought to be based on an egocentric reference system which relates vector information derived from path integration to views of landmarks experienced en route and at the goal. Here we show that honeybees also possess an allocentric form of spatial memory which allows localization of multiple places relative to the intended goal, the hive. The egocentric route memory, which is called the specialized route memory (SRM) here, initially dominates navigation when an animal is first trained to a feeding site and then released at an unexpected site and this is why it is the only reference system detected so far in experiments with bees. However, the SRM can be replaced by an allocentric spatial memory called the general landscape memory (GLM). The GLM is directly accessible to the honeybee (and to the experimenter) if no SRM exists, for example, if bees were not trained along a route before testing. Under these conditions bees return to the hive from all directions around the hive at a speed comparable to that of an equally long flight along a trained route. The flexible use of the GLM indicates that bees may store relational information on places, connections between landmarks and the hive and/or views of landmarks from different directions and, thus, the GLM may have a graph structure, at least with respect to one goal, i.e. the hive.     

Analysis of three overexpression systems for VanX, the Zinc(II) dipeptidase required for high-level vancomycin resistance in bacteria

The gene from Enterococcus faecilis encoding the dipeptidase VanX was subcloned into overexpression vectors pET-5b, pET-27b, and IMPACT-T7, and VanX was overexpressed in BL21(DE3) pLysS Escherichia coli. The pET-5b-vanx overexpression plasmid produces VanX at approximately 12 mg/L under optimum conditions. VanX produced from this overexpression system exists primarily as a dimer in solution, binds ca. 1 Zn(II) ion per monomer, and exhibits K(m) and k(cat) values of 500 +/- 40 microM and 0.074 +/- 0.001 s(-1), respectively, when l-alanine-p-nitroanilide is used as substrate. The IMPACT-T7-vanx overexpression plasmid produces a VanX-fusion protein with a chitin-binding domain that allows for purification of the fusion construct with a chitin column. Cleavage of the fusion protein is completed by an on-column chemical cleavage, resulting in approximately 10 mg/L of purified VanX. VanX produced from this system is identical to that produced from the pET-5b system, except the CD spectrum of the IMPACT-T7-produced VanX suggests a small change in secondary structure. This change in secondary structure does not affect any of the kinetic or metal-binding properties of the enzyme. The pET-27b-vanx overexpression plasmid produces and secretes VanX into the growth medium; this system allows for 20 mg of VanX to be isolated per liter of growth medium. The pET-27b-produced VanX is identical to that produced from pET-5b.