Sliding motion modulates stiffness and friction coefficient at the surface of tissue engineered cartilage.

OBJECTIVE: Functional cartilage tissue engineering aims to generate grafts with a functional surface, similar to that of authentic cartilage. Bioreactors that stimulate cell-scaffold constructs by simulating natural joint movements hold great potential to generate cartilage with adequate surface properties. In this study two methods based on atomic force microscopy (AFM) were applied to obtain information about the quality of engineered graft surfaces. For better understanding of the molecule-function relationships, AFM was complemented with immunohistochemistry. METHODS: Bovine chondrocytes were seeded into polyurethane scaffolds and subjected to dynamic compression, applied by a ceramic ball, for 1h daily [loading group 1 (LG1)]. In loading group 2 (LG2), the ball additionally oscillated over the scaffold, generating sliding surface motion. After 3 weeks, the surfaces of the engineered constructs were analyzed by friction force and indentation-type AFM (IT-AFM). Results were complemented and compared to immunohistochemical analyses. RESULTS: The loading type significantly influenced the mechanical and histological outcomes. Constructs of LG2 exhibited lowest friction coefficient and highest micro- and nanostiffness. Collagen type II and aggrecan staining were readily observed in all constructs and appeared to reach deeper areas in loaded (LG1, LG2) compared to unloaded scaffolds. Lubricin was specifically detected at the top surface of LG2. CONCLUSIONS: This study proposes a quantitative AFM-based functional analysis at the micrometer- and nanometer scale to evaluate the quality of cartilage surfaces. Mechanical testing (load-bearing) combined with friction analysis (gliding) can provide important information. Notably, sliding-type biomechanical stimuli may favor (re-)generation and maintenance of functional articular surfaces and support the development of mechanically competent engineered cartilage.

Fatal outcome of a hepatitis B virus transfusion-transmitted infection.

BACKGROUND AND OBJECTIVES: In 2008, hepatitis B virus (HBV) DNA testing was not yet mandatory for the screening of blood donations in Switzerland. At that time, HBsAg was the only specific mandatory marker for HBV. The importance of high sensitivity for HBV NAT screening is shown. MATERIALS AND METHODS: Donor and recipient of a transfusion-transmitted HBV infection were followed up. Multiple samples were tested for HBV serological and molecular markers. RESULTS: At donation, the donor appeared healthy, HBsAg was negative and had a normal ALAT level. Ten weeks later, clinical symptoms suggested acute HBV infection as was confirmed with positive HBsAg, HBeAg, anti-HBc IgG, anti-HBc IgM and anti-HBe. The archived sample from the original donation was negative for anti-HBc, but positive for HBV DNA (17 IU/ml). A recipient transfused with the red cell concentrate was HBV DNA positive (3100 IU/ml) 3 months post-transfusion. After five months, HBsAg, HBeAg, anti-HBc and HBV DNA (1.1 x 10(11) IU/ml) were positive. Two weeks later, the patient died from complications associated with HBV infection and his underlying bone marrow disease. CONCLUSIONS: The present case illustrates the importance of introducing highly sensitive HBV NAT screening strategy to prevent possible HBV transfusion-transmitted infections from donors with low viral load.

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps.

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.

Pulsatile dynamic stiffness of cartilage-like materials and use of agarose gels to validate mechanical methods and models.

Stiffness is a fundamental indicator of the functional state of articular cartilage. Reported test modes include compressive incremental strain to determine the equilibrium modulus, and sinusoidal strain to determine the dynamic modulus and stress/strain loss angle. Here, initial development is described for a method recognizing that gait is pulsatile. Agarose gels have been used by others for validation or comparison of mechanical test methods and models for cartilage and proteoglycan aggregate. Accordingly, gels ranging from 0.5 to 20% agarose were prepared. Pulsatile stiffness in both indentation and unconfined compression were closely reproducible. Stiffness as a function of agarose concentration rose exponentially, as found using other methods. Indentation stiffness was higher than for unconfined compression and ranged from approximately 2.0 kPa for 0.5% gel to approximately 3,800 kPa for 20% gel. Pulsatile dynamic stiffness appears to be a useful method, although further development is needed. Agarose gel stiffness values obtained by other methods were reviewed for comparison. Unfortunately, reported values for a given agarose concentration ranged widely (e.g. fourfold) even when test methods were similar. Causes appear to include differences in molecular weight and gel preparation time-temperature regimens. Also, agarose is hygroscopic, leading to unintended variations in gel composition. Agarose gels are problematic materials for validation or comparison of cartilage mechanical test methods and models.

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.

The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop.

A histidine residue with a pKa of 7 has been inferred to act as a general acid-base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochondrial isoenzyme Mib-CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X-ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small-angle X-ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild-type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters K(m) or Vmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid-base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.

The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.

Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.

The DNA-binding properties of an artificial 42-residue polypeptide derived from a natural repressor.

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-1, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.

Molecular and pharmacological characterization of the human CCKB receptor.

The human cholecystokinin B (CCKB) receptor has been isolated from a human temporal cortex cDNA library. Transient transfection of the receptor into COS-M6 cells resulted in high specific binding of 125I-sulphated CCK-8 labelled with Bolton and Hunter Reagent (KD = 31 pM). Competition experiments yielded the expected CCKB receptor ligand binding profile for agonists and antagonists. Similar results were obtained in human small cell lung carcinoma cells, which express an endogenous CCKB receptor. Extensive functional characterization of the receptor was performed in stably transfected HeLa cells using intracellular calcium imaging and microphysiometry techniques. Molecular analysis of the human CCKB receptor using Southern blotting of genomic DNA suggests the presence of a single gene for the CCKB receptor with no closely related homologues. This was confirmed by the polymerase chain reaction cloning of identical receptor coding sequences from human small cell lung carcinoma cells and human gastric enterochromaffin-like cell-oma (ECLoma) tissue.

Adenosine triphosphate and arachidonic acid stimulate glycogenolysis in primary cultures of mouse cerebral cortical astrocytes.

Adenosine triphosphate (ATP) promotes glycogenolysis in primary cultures of mouse cerebral cortical astrocytes with an EC50 of 1.5 microM. A pharmacological analysis indicates an involvement of purinergic P2Y receptors in this action of ATP. Application of either arachidonic acid (AA), or certain unsaturated fatty acids, also results in glycogen breakdown. The EC50 of AA is approximately 50 microM. Thus ATP and AA can be added to the list of neuroactive agents that control glycogen levels in astrocytes, which includes noradrenaline, vasoactive intestinal peptide (VIP), adenosine and histamine.

Purification, characterization and N-terminal sequence of phosphoserine aminotransferase from the green alga Scenedesmus obliquus, mutant C-2 A'.

Phosphoserine aminotransferase (EC 2.6.1.52), an enzyme of the "phosphorylated pathway" leading to the formation of serine, was purified from Scenedesmus obliquus, mutant C-2 A'. Purification started from the soluble supernatant of a crude cell homogenate and included different affinity and DEAE chromatographic techniques, as well as gel filtration. The purified phosphoserine aminotransferase was enriched 1537-fold and identified to be a homodimer with subunit molecular masses of 40 kDa, each. The absorption spectrum is consistent with the presence of pyridoxal-5-phosphate as cofactor. From the purified enzyme 18 amino acids of the N-terminus could be determined, showing at least 67% homology with the serC gene encoding phosphoserine aminotransferases from bacterial organisms.

The risk of a second diagnostic window with 4th generation HIV assays: Two cases.

BACKGROUND: Despite the improved sensitivity of the 4th generation combined antigen/antibody HIV assays, detection of HIV in the early phase of an infection may still be ineffective. OBJECTIVES: Description of two cases that highlight the existence of the "second diagnostic window phase" observed with commonly used sensitive 4th generation HIV assays. STUDY DESIGN: Samples were screened with different 4th generation HIV assays. HIV infection was confirmed with an HIV I/II antibody assay, a HIV-1 p24 antigen assay, the INNO-LIA HIV I/II Score Line immunoassay and HIV-1 PCR. RESULTS: In both investigated cases, the limitations of the 4th generation HIV assays within the second diagnostic window were apparent. CONCLUSIONS: The overall sensitivity of the commercial 4th generation HIV assays is currently higher than the 3rd generation HIV assays. Nevertheless, the rare occurrence of a second diagnostic window with 4th generation HIV assays strongly suggests that the following up testing algorithms need to be adjusted accordingly.

Purification, metal cofactor, N-terminal sequence and subunit composition of a 5-aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'.

5-Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C-2A', starting with serial affinity chromatography according to Wang et al., followed by separation on DEAE-Cellulose DE 52, TSKgel Toyopearl HW-55 and FPLC on Mono Q. The enzyme was purified 117-fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 microkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N-terminus could be determined, showing a moderate similarity to 5-aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282+/-5 kDa. 42+/-1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six-membered ring. Additionally, there is some evidence that two of the rings form a sandwich-like complex.

Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of N-terminal binding epitope in MM-CK and BB-CK.

The molecular origin of the isoenzyme-specific interaction of cytosolic creatine kinase isoenzymes, muscle-type creatine kinase and brain-type creatine kinase, with myofibrillar structures has been studied by confocal microscopy in an functional in situ binding assay with chemically skinned, unfixed skeletal muscle fibers using wild-type and chimeric creatine kinase isoproteins. The specific interaction of both wild-type isoforms with the sarcomeric structure resulted in a stable, isoform-characteristic labeling pattern with muscle-type creatine kinase bound exclusively and tightly to the sarcomeric M-band while brain-type creatine kinase was confined to the I-band region. Chimeric proteins of both muscle-type and brain-type creatine kinases were constructed to localize the corresponding binding domain(s). Exchanged domains included the N-terminal part (residues 1-234), the region containing an isoenzyme 'diagnostic box' (residues 235-285) and the C-terminal part (residues 286-380). The purified recombinant proteins were all fully intact and enzymatically active. All chimeric proteins containing the N-terminal region (amino acid 1-234) of muscle-type or brain-type creatine kinase were always specifically targeted to the sarcomeric M-band or I-band, respectively. We therefore propose that the relevant epitope(s), determining the isoenzyme-specific targeting in skeletal muscle, are entirely located within the N-terminal regions of both cytosolic creatine kinase isoforms.

Hepatitis C virus-polymerase chain reaction minipool testing: 3 years in the largest Swiss blood transfusion service.

BACKGROUND AND OBJECTIVES: Hepatitis C virus-polymerase chain reaction (HCV-PCR) minipool testing can improve the safety of labile blood products owing to a reduction in the diagnostic preseroconversion window period. In Switzerland, HCV-PCR minipool testing for the release of labile blood components became mandatory in September 1999. In the largest Swiss blood transfusion centre, HCV-PCR minipool testing began in January 1999. This report analyses the performance of the test during a 3-year period: 1 January 1999 to 31 December 2001. MATERIALS AND METHODS: EDTA-blood was collected in either standard tubes or plasma preparation (PPT) tubes from 10 blood transfusion services in Switzerland and then sent to the Blood Transfusion Service SRC Berne. Up to 48 donor samples were pooled overnight using Tecan Genesis RSP 200/8 pipettors. Viral RNA was extracted by using the Qiagen QIAamp 96 viral RNA BioRobot kit on a BioRobot 9604. For PCR amplification and detection of HCV or internal control (IC) sequences, the Roche Cobas Amplicor v2.0 test kit was used. Data management, pool resolution and identification of positive samples were performed using the PMS Software from Tecan. RESULTS: In the 3-year period from 1 January 1999 to 31 December 2001, 839056 blood donor samples were tested in minipools of up to 48 samples. Thirty-five HCV-PCR-positive donations were identified. Thirty-four samples had antibodies against HCV and were therefore also detected by screening for antibody to HCV (anti-HCV). In October 2001, one seronegative (but PCR-positive) donor was detected. CONCLUSIONS: HCV-PCR minipool testing was successfully introduced in the largest Swiss blood transfusion service. It was shown that the release of HCV-PCR minipool results can be accomplished concurrently with the results of serological analysis. The challenge with a seronegative, but PCR-positive, donor demonstrates that the minipool testing strategy adds additional safety to blood products.

[Simultaneous radiochemotherapy of locally advanced stage T4 breast cancer]. / Simultane Radio-Chemotherapie des lokal fortgeschrittenen Mammakarzinoms im Stadium T4.

The local efficacy of simultaneous radiochemotherapy was tested in 17 women with 18 carcinomas of the breast in stage T4 only. Treatment was conducted for a period of 6 months with 6 courses of drug therapy (A + E) coordinated simultaneously with 6 courses of radiotherapy. In 72% of the cases we found considerable (greater than 80%) or complete (100%) regression of the tumour as a result of this combined treatment. The median survival time is 35 months.

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity.

Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base.

Monitoring biomolecular interactions by time-lapse atomic force microscopy.

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.