The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model.

OBJECTIVE: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. METHODS: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. RESULTS: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. CONCLUSIONS: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Establishment of immortalized periodontal ligament progenitor cell line and its behavioural analysis on smooth and rough titanium surface.

Periodontal ligament (PDL) can be obtained from patients undergoing orthodontic treatment. PDL contains progenitor cells that can be expanded and differentiated towards several mesenchymal lineages in vitro. Furthermore, PDL-derived cells have been shown to generate bone- and PDL-like structures in vivo. Thus, PDL cells, combined with suitable biomaterials, represent a promising tool for periodontitis-related research and PDL engineering. Here, a new PDL cell line using lentiviral gene transfer of human telomerase reverse transcriptase (hTERT) was created. HTERT-expressing PDL cells showed similar morphology and population doubling time but an extended lifespan compared to the primary cells. In addition, PDL-hTERT cells expressed several characteristic genes and upon osteogenic stimulation produced a calcified matrix in vitro. When cultivated on two topographically different titanium scaffolds (MA and SLA), PDL-hTERT cells exhibited augmented spreading, survival and differentiation on smooth (MA) compared to rough (SLA) surfaces. These findings differ from previously reported osteoblast behaviour, but they are in agreement with the behaviour of chondrocytes and gingival fibroblasts, suggesting a very cell type-specific response to different surface textures. In summary, we report the testing of titanium biomaterials using a new PDL-hTERT cell line and propose this cell line as a useful model system for periodontitis research and development of novel strategies for PDL engineering.

A force-based protein biochip.

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Force spectroscopy with single bio-molecules.

For many biological molecules, force is an important functional and structural parameter. With the rapidly growing knowledge about the relation between structure, function, and force, single-molecule force spectroscopy has become a versatile analytical tool for the structural and functional investigation of single bio-molecules in their native environments. Within the past year, detailed insights into binding potentials of receptor ligand pairs, protein folding pathways, molecular motors, DNA mechanics and the functioning of DNA-binding agents (such as proteins and drugs), as well as the function of molecular motors, have been obtained.

Mechanical stability of single DNA molecules.

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (

Characterization of IgG Langmuir-Blodgett films immobilized on functionalized polymers.

The bioactivity of anti-human IgG Langmuir-Blodgett (LB) films, the non-specific adsorption of protein and the topography of anti-IgG LB films have been studied for application in immunosensors. The antibody (AB) LB films were horizontally deposited on glass and functionalized polymers, such as carboxy-poly(vinyl chloride) (PVC-COOH), chloropropyl and aminopropyl sol-gel. The LB films were characterized by means of ellipsometry, atomic force microscopy (AFM) and bicinchoninic acid (BCA) protein test. The interpretation of ellipsometric data was performed using a one-layer model. Non-specifically adsorbed protein was desorbed by washing the IgG film in 0.5 M NaCl, 2 M NaCl and 1% N-cetyl-N,N,N-trimethylammoniumbromide detergent solution resulting in a 50% reduction of the film thickness. The mean thickness of an anti-IgG film on glass measured by ellipsometry, PVC-COOH and aminopropyl sol-gel was 9+/-2, 11+/-1 and 23+/-8 nm, respectively. According to the BCA test 6-8 mug antibody (AB) per slide was bound to the functionalized polymers, but only 3 mug AB per slide was adsorbed on glass. The average distance of anti-IgG granules as indicated by AFM measurements on PVC-COOH, chloropropyl and aminopropyl sol-gel was 42+/-20, 34+/-3 and 23+/-4 nm. The average distance of granular AB structures on glass, however, was 150+/-50 nm.

Sequence-dependent mechanics of single DNA molecules.

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.

How strong is a covalent bond?

The rupture force of single covalent bonds under an external load was measured with an atomic force microscope (AFM). Single polysaccharide molecules were covalently anchored between a surface and an AFM tip and then stretched until they became detached. By using different surface chemistries for the attachment, it was found that the silicon-carbon bond ruptured at 2.0 +/- 0.3 nanonewtons, whereas the sulfur-gold anchor ruptured at 1.4 +/- 0.3 nanonewtons at force-loading rates of 10 nanonewtons per second. Bond rupture probability calculations that were based on density functional theory corroborate the measured values.

Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2.

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.

Direct detection of domains in phospholipid bilayers by grazing incidence diffraction of neutrons and atomic force microscopy.

The geometry of domains in phospholipid bilayers of binary (1:1) mixtures of synthetic lecithins with a difference in chain length of four methylene groups has been studied by two independent, direct and complementary methods. Grazing incidence diffraction of neutrons provided gel domain sizes of less than 10 nm in both the gel and the coexistence phase of the mixture, while no domains were detected for the fluid phase. For the coexistence region, the neutron data suggest that domains grow in number rather than in size with decreasing temperature. Atomic force microscopy was used to study gel phase size and shape of the domains. The domains imaged by atomic force microscopy exhibit a rather irregular shape with an average size of 10 nm, thus confirming the neutron results for this phase. The good agreement between atomic force microscopy and neutron results, despite the completely different nature of their observables, has potential for the future development of refined models for the interpretation of neutron data from heterogeneous membranes in terms of regularly spaced and spatially extended scatterers.

Autism in immigrants: children born in Sweden to mothers born in Uganda.

Three boys diagnosed as suffering from autistic disorder were born in Sweden to mothers born in Uganda. Two were related but the third boy was unrelated to the others. The prevalence for autistic disorder in Göteborg children born to mothers who were born in Uganda was 15% which is almost 200 times higher than in the general population of children. The possible reason for the high autism rate in this particular ethnic subgroup is discussed.

Is autism more common now than ten years ago?

A total population study of children, aged 13 years and under, suggested that there has been an apparent rise in the frequency of autistic disorder and autistic-like conditions (excluding Asperger's syndrome) in one area of western Sweden over the last ten years. The frequency was 4.0/100,000 in 1980, 7.5/10,000 in 1984 and 11.6/10,000 in 1988 in the city of Göteborg. Even though the prevalence rates refer to slightly different age cohorts, it was concluded that the apparent increase is in part due to better detection, but also to new cases born to immigrant parents. Typical cases of autistic disorder accounted for 75% of cases, and 20% had normal or near-normal IQs.

Autism under age 3 years: a clinical study of 28 cases referred for autistic symptoms in infancy.

Twenty-eight children referred with a preliminary diagnosis of autistic disorder under age 3 yrs were extensively examined from the neuropsychiatric point of view and followed up for several months to several years. A diagnosis of autistic disorder was confirmed in 75% of the cases. A variety of associated medical conditions was identified. It was concluded that autism can be diagnosed in a substantial proportion of cases before age 3 yrs and that the neurobiological background is similar to that seen in older autistic children.

Epilepsy presenting as infantile autism? Two case studies.

Two cases diagnosed as suffering from infantile autism are described. Underlying epileptogenic changes in the EEG were not disclosed until relatively late in the course of the psychiatric disorder. Anticonvulsive pharmacological treatment led to complete disappearance of psychotic symptoms and to simultaneous disappearance of the pathological EEG changes. Alternative mechanisms are proposed to account for the clinical phenomena.

Social class and infantile autism.

Twenty infantile autistic children, constituting what is likely to be the majority of the total population of autistic children born in the years 1962 through 1973 and living in Göteborg, Sweden, by the end of 1978, were compared with a random population sample of 59 7-year-old Göteborg children with regard to social class. Two different social classification systems were used, one that takes account only of the father's occupation and one that includes several other parameters. The distributions of social class were almost identical in the infantile autism group and in the random group. With respect to some other social circumstances the two groups were very similar. Thus, the present results lend no support for the view that autistic children tend to come from high social classes.

Infantile autism and puberty.

While the prognosis for autistic children is generally poor, some show substantial improvement during childhood. In a Swedish sample of five cases deterioration or severe symptom aggravation at the onset of puberty is described that followed earlier improvement. Autistic children with pubertal deterioration may constitute a meaningful subgroup of the syndrome. The matter deserves more attention in future follow-up studies.

[Efficiency of patients with chronic renal failure (author's transl)]. / Die Leistungsfähigkeit von chronisch Niereninsuffizienten im Stadium der kompensierten Retention.

In 77 patients with chronic renal failure - all were conservative treated - exercise-tests were performed with the bicycle and in 67 cases spiro-ergometric tests. We found a correlation between the decrease of hemoglobin and the decrease of efficiency and a linear function between the decrease of hemoglobin and the increase of creatinin in serum. There was no direct correlation between the increase of creatinin and the decrease of efficiency. The efficiency, measured in Watt, and the oxygen pulse correlated. More patients with hypertonia exceeded the level of 70 of the quotient heart volume/oxygen pulse but those with normal pressure. The efficiency was reduced in general: only 13,4% obtained 100 Watt, 28,4% 75 Watt and the rest of 58,2% were of bad efficiency and performed only 50 Watt or less.