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Temperature scanning FTIR analysis of hydrogen bonding states of various saccharides in amorphous matrixes below and above their glass transition temperatures.

Temperature scanning Fourier transform infrared, TS-FTIR, spectroscopy of various amorphous sugar matrixes was conducted to investigate the relationship between the glass transition temperature, T(g), of an amorphous sugar matrix and the nature of the hydrogen bonds in the matrix. An amorphous sugar matrix was prepared by air-drying an aqueous solution of sugar, and the degree of formation of hydrogen bonds in the matrix was evaluated at different temperatures using the peak positions of the IR band corresponding to the O-H stretching vibration at around 3400 cm(-1). The T(g) value increased with increasing peak position of the O-H stretching vibration at T(g) and were correlated reasonably well with the magnitude of the peak shift by the temperature increase (from 25 degrees C) to the T(g) value. This demonstrates that the amorphous sugar matrix, in which the segments are fixed by fewer hydrogen bonds, has a higher thermal resistance. The glycosidic linkage largely contributes to the restriction of the segments, pyranose ring, rather than a hydrogen bond. As the degree of polymerization of pyranose rings increases, the degree of hydrogen bond formation needed to hold the matrix in a fixed position decreases. However, the magnitude of the restriction of pyranose rings by a glycosidic linkage changes depending on the type: the restrictions imposed by alpha-1,1 and -1,6 glycosidic linkages are the tightest and most flexible of all of the types of glycosidic linkages, respectively.

Hydrogen Bonding↗

A matrix-located processing peptidase of plant mitochondria.

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.

Cell Nucleus↗

Impact of pharmacokinetic-pharmacodynamic model linearization on the accuracy of population information matrix and optimal design.

Influence of experimental design on hyperparameter estimates precision when performing a population pharmacokinetic-pharmacodynamic (PK-PD) analysis has been shown by several studies and various approaches have been proposed for optimizing or evaluating such designs. Some of these methods rely on the optimization of a suitable scalar function of the population information matrix. Unfortunately for the nonlinear models encountered in pharmacokinetics or pharmacodynamics the latter is particularly difficult to evaluate. Under some assumptions and after a linearization of the PK-PD model a closed form of this matrix can be obtained which considerably simplifies its calculation but leads to an approximation. The aim of this paper is to evaluate the quality of the latter and its potential impact, when comparing or optimizing population designs and to relate it to Bates and Watts curvature measures. Two models commonly used in PK-PD were considered and nominal hyperparameter values when chosen for each one. Several population designs were studied and the associated population information matrix was computed for each using the approximate procedure and also using a reference method. Design optimizations were calculated under constraints for each model from the reference and approximate population information matrix. Nonlinearity curvatures were also computed for every model and design. The impact of model linearization when calculating the population information matrix was then examined in terms of lower bound accuracies on the hyperparameter estimates, design criterion variation, as well as D-optimal population designs, these results being related to nonlinearity curvature measures. Our results emphasize the influence of the parameter effects curvature when deriving the lower bounds of the hyperparameter estimates precision for a given design from the approximate population information matrix especially for hyperparameters quantifying the PK-PD interindividual variability. No discrepancies were detected between the population D-optimal designs obtained from the approximate and reference matrix despite some minor differences in criterion variation with respect to the design. More pronounced differences were, however, observed when comparing the amplitudes of criterion variation which can lead to errors when calculating design efficiencies. From a practical point of view, a strategy easily applicable by the pharmacokineticist for avoiding such problems in the context of population design optimization or comparison is then proposed.

Humans↗

Actively transcribed genes are associated with the nuclear matrix.

In the chicken oviduct, it has been well documented that steroid hormones stimulate the transcription of specific genes such as the ovalbumin gene. In addition to the presence of specific hormone receptors in the tissue, gene expression seems to require that target genes exist in large DNase I sensitive chromosomal domains. This structure appears necessary but not sufficient for transcriptional activation. In search of still other levels of control, we have investigated the interactions of genes with the nuclear matrix, a structure which has been implicated in DNA synthesis, transcription and RNA processing. Here we have isolated nuclear matrix and used a nondegradative method to fractionate nuclear DNA based on its preferential association with the matrix. The preparation was digested with a restriction enzyme and both matrix-bound and released DNAs were recovered. We found that only actively expressed genes were associated with the matrix. Furthermore, within a 100-kilobase (kb) DNase I sensitive chromosomal domain, only the transcribed regions were associated with the matrix. This association was shown to be reversible when hormone was withdrawn. Our results suggest that the nuclear matrix is the site of nuclear transcription and may represent another potential level of control for regulation of gene expression in the eukaryotic cell.

Animals↗

Automated screening for genomic imbalances using matrix-based comparative genomic hybridization.

Genome-wide screening for chromosomal imbalances using comparative genomic hybridization (CGH) revealed a wealth of data on previously unrecognized tumor-specific genomic alterations. CGH to microarrays of DNA, an approach termed matrix-CGH, allows detection of genomic imbalances at a much higher resolution. We show that matrix CGH is also feasible from small tissue samples requiring universal amplification of genomic DNA. Because widespread application of matrix-CGH experiments using large numbers of DNA targets demands a high degree of automation, we have developed a protocol for a fully automated procedure. The use of specialized instrumentation for the generation of DNA chips, their hybridization, scanning, and evaluation required numerous alterations and modifications of the initial protocol. We here present the elaboration and testing of automated matrix-CGH. A chip consisting of 188 different genomic DNA fragments, cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors and immobilized in replicas of 10, was used to assess the performance of the automated protocol in determining the gene dosage variations in tumor cell lines COLO320-HSR, HL60, and NGP. Although ratios of matrix-CGH were highly concordant with results of chromosomal CGH (85%), the dynamic range of the matrix-CGH ratios was highly superior. Investigation of the two amplicons on 8q24 in COLO320-HSR and HL60, containing the MYC gene, revealed a homogeneous amplicon in COLO320-HSR but a heterogeneous amplification pattern in HL60 cells. Although control clones for normalization of the signal ratios can be predicted in cases with defined chromosomal aberrations, in primary tumors such data are often not available, requiring alternative normalization algorithms. Testing such algorithms in a primary high-grade B-cell lymphoma, we show the feasibility of this approach. With the matrix-CGH protocol presented here, robust and reliable detection of genomic gains and losses is accomplished in an automated fashion, which provides the basis for widespread application in tumor and clinical genetics.

Chromosome Aberrations↗

The regulation of the oxidation of fatty acids and other substrates in rat heart mitochondria by changes in the matrix volume induced by osmotic strength, valinomycin and Ca2+.

1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 microliter/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 microliter/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 microliter/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria [Halestrap & Dunlop (1986) Biochem. J. 239, 559-565]. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30 s at 30 degrees C and with K0.5 for Ca2+ of 0.3 microM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37 degrees C in the presence of 0.65 microM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181 +/- 0.029 microliter/mg of protein (n = 7, P less than 0.001), similar to the Ca2+-induced changes observed with liver mitochondria [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.

Animals↗

Compartmentalization of the matrix formed by nucleus pulposus and annulus fibrosus cells in alginate gel.

Intervertebral disc cells cultured in alginate gel are capable of reforming in alginate, a matrix that consists of two compartments: a rim of metabolically active cell-associated matrix and a more abundant, but metabolically less active, further removed matrix. At any one age and in most species, the cell-associated matrix formed by a nucleus pulposus or annulus fibrosus cell cultured in this way is less abundant than that formed by an articular chondrocyte. In both the cell-associated matrix and further removed matrix, the ratio of aggrecan to collagen is significantly higher in the case of nucleus pulposus than of annulus fibrosus, a feature that also distinguishes the matrices of the nucleus pulposus and annulus fibrosus in vivo. Nucleus pulposus and annulus fibrosus cells from older donors show a decreased ability to reform a cell-associated matrix rich in aggrecan. There is, however, some evidence that gene therapy and/or exposure of the cells to defined stimulatory factors can help overcome some of these age-related limitations. This contention is supported by recent evidence that nucleus pulposus and annulus fibrosus cells from adult donors can be manipulated to form, using the recently developed alginate-recovered chondrocyte system, a resilient tissue that bears many of the characteristics of the tissue in which these cells reside in vivo.

Alginates↗

Dermal regeneration in native non-cross-linked collagen sponges with different extracellular matrix molecules.

Collagenous dermal templates can prevent scarring and wound contraction in the healing of full-thickness defects. In a porcine wound model, full-thickness wounds were substituted by reconstituted and native collagen sponges in combination with autologous split-skin mesh grafts and covered with a semipermeable wound membrane. Native collagen sponges were also linked with either hyaluronic acid, elastin, or fibronectin. Reconstituted collagen matrixes, composed of cross-linked small collagen fibrils, disintegrated within a week and did not contribute to dermal regeneration, whereas native collagen matrixes, composed of intact collagen fibers, disintegrated within 2 weeks and did contribute to dermal regeneration. Addition of extracellular matrix proteins retarded the disintegration to 4 weeks. However, fibronectin-treated matrixes caused aberrant epithelization. When hyaluronic acid was added, matrixes were invaded by more fibroblasts and myofibroblasts. This process correlated with fibrosis and wound contraction. In contrast, the native collagen/elastin matrix reduced the amount of fibroblasts and myofibroblasts. This latter matrix resulted in optimal dermal regeneration and little wound contraction.

Journal Article↗

Wound fluids from saline solution- and Staphylococcus aureus peptidoglycan-inoculated sponges induce expression of matrix metalloproteinase 13 messenger ribonucleic acid by cultured rat fibroblasts.

Polyvinyl alcohol sponges inoculated with Staphylococcus aureus peptidoglycan induce an accelerated wound healing response when implanted subcutaneously in rats. S. aureus peptidoglycan leads to a marked increase (50%) in reparative tissue collagen (as measured by hydroxyproline) by 4 days. However, this effect drops by 7 days and by 14 days; hydroxyproline levels are similar in sponges inoculated with S. aureus peptidoglycan or saline solution. These data suggest a very active early remodeling process in S. aureus peptidoglycan sponge reparative tissue. Consistent with this observation, we had found that steady-state levels of matrix metalloproteinase-13 mRNA were higher and persisted longer in S. aureus peptidoglycan sponge reparative tissue than in controls. We hypothesized that S. aureus peptidoglycan might induce a change in reparative tissue fibroblast phenotype or modify the character of the wound fluid. Fibroblasts obtained from saline solution- and S. aureus peptidoglycan-inoculated sponges 4 days after subcutaneous implantation and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum were similar with respect to morphologic features, proliferation, and expression of pro alpha1 (I) and alpha1 (III) collagens and tissue inhibitor of metalloproteinase-1 mRNA by Northern blot analysis. Neither cell type expressed matrix metalloproteinase-13 mRNA. No changes in the above parameters were detected when such fibroblasts were cultured for 24 hours in the presence of 0.5 mg of S. aureus peptidoglycan per 10 ml of medium or with fluid obtained from control sponges cultured for 12 hours with phosphate-buffered saline solution. Wound fluids extracted with Eagle's minimal essential medium by homogenization of saline solution- and S. aureus peptidoglycan-inoculated sponges implanted subcutaneously for 12 hours did not affect the proliferation of the fibroblasts. However, the extracts had a profound effect on the cellular expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase-13, and pro alpha1 (I) collagen mRNA. Specifically, expression of matrix metalloproteinase-13 mRNA was induced, expression of pro alpha1 (I) collagen mRNA was reduced by 70%, and expression of tissue inhibitor of metalloproteinase-1 mRNA was increased by 150%. These changes were the same irrespective of whether the wound fluid was obtained from saline solution- or S. aureus peptidoglycan-inoculated sponges. Fluid obtained from S. aureus peptidoglycan-inoculated sponges, which contain a greater inflammatory exudate than saline solution-inoculated sponges do, is enriched in matrix metalloproteinase-13 mRNA-inducing activity. The nature of the factor(s) that induces matrix metalloproteinase-13 mRNA expression is not known. However, preliminary data suggest that the matrix metalloproteinase-13-inducing factor(s) is heterogeneous with regard to size and is temperature sensitive and trypsin resistant.

Journal Article↗

Effect of calcium on structure and function of a hyaluronic acid matrix: carbon-13 nuclear magnetic resonance analysis and the diffusional behavior of small solutes.

Natural-abundance (13)C NMR at 25.16 MHz has been used to study a 2.5% matrix of hyaluronic acid at various degrees of polymerization and at various ionic strengths. Peak assignment is facilitated by comparing proton-decoupled and off-resonance-decoupled spectra of a hyaluronidase-depolymerized matrix with spectra from relevant monosaccharides. In contrast to the spectrum following depolymerization, the spectrum for intact matrix has considerable broadening, particularly for peaks assigned to the N-acetylglucosamine moiety. This is most dramatic for the hydroxymethylene carbon. With the addition of Ca(2+) above 5 mM these broadened peaks narrow and approach the sharpness observed for the hyaluronidase digest. There is no shift in resonance peak positions. These changes are quantitatively less impressive if Na(+) is substituted for Ca(2+). The data suggest the existence of a considerable degree of order in regions of the matrix at physiological concentrations of Ca(2+). Within such a matrix the translational movement of lysine and glucose is enhanced relative to that in a matrix of agarose. Further addition of Ca(2+) abrogates not only matrix order, but the enhanced diffusivity as well.

Calcium↗

Purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro.

One of the major structural proteins of vesicular stomatitis virus is a small, nonglycosylated, matrix protein which associates with the nucleocapsid core during final stages of morphogenesis and budding. Biochemical and genetic studies suggested that the matrix protein regulates RNA synthesis both in vitro and in vivo. We have purified biologically active matrix protein from the virus and have directly shown that it significantly inhibits RNA synthesis in vitro mediated by the virion-associated RNA polymerase at low ionic strength (0.02 M). The inhibition was greater than 80% when the ratio of matrix protein to the major nucleocapsid protein in the transcribing complex was 2:1 (wt/wt). The inhibition was found to be at the level of RNA chain elongation and not at the initiation step. Electron microscopic studies revealed that inhibition of transcription by matrix protein was accompanied by a profound structural change of the transcribing nucleocapsid from an extended structure to a highly compact form. At higher ionic strength (0.12 M), the matrix protein failed to interact with the nucleocapsid. The matrix protein appears to be involved in condensing the nucleocapsid and blocking transcription during maturation of the virus particle.

Gene Expression Regulation↗

Distinct presynaptic control of dopamine release in striosomal and matrix areas of the cat caudate nucleus.

By use of a sensitive in vitro microsuperfusion method, the cholinergic prsynaptic control of dopamine release was investigated in a prominent striosome (areas poor in acetylcholinesterase activity) located within the core of cat caudate nucleus and also in adjacent matrix area. The spontaneous release of [3H]dopamine continuously synthesized from [3H]tyrosine in the matrix area was found to be twice that in the striosomal area; the spontaneous and potassium-evoked releases of [3H]dopamine were calcium-dependent in both compartments. With 10(-6) M tetrodotoxin, 5 x 10(-5) M acetylcholine stimulated [3H]dopamine release in both striosomal and matrix areas, effects completely antagonized by atropine (10(-6) M), thus showing the involvement of muscarinic receptors located on dopaminergic nerve terminals. Experiments without tetrodotoxin revealed a more complex regulation of dopamine release in the matrix: (i) In contrast to results seen in the striosome, acetylcholine induced only a transient stimulatory effect on matrix dopamine release. (ii) Although 10(-6) M atropine completely abolished the cholinergic stimulatory effect on [3H]dopamine release in striosomal area, delayed and prolonged stimulation of [3H]dopamine release was seen with atropine in the matrix. The latter effect was completely abolished by the nicotinic antagonist pempidine (10(-5) M). Therefore, in the matrix, in addition to its direct (tetrodotoxin-insensitive) facilitatory action on [3H]dopamine release, acetylcholine exerts two indirect (tetrodotoxin-sensitive) opposing effects: an inhibition and a stimulation of [3H]dopamine release mediated by muscarinic and nicotinic receptors, respectively.

Acetylcholine↗

Dynamics, structure, and function are coupled in the mitochondrial matrix.

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

Animals↗

A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization.

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

Amino Acid Sequence↗

Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix.

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Delta cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.

Carrier Proteins↗

Organic matrix composition and ultrastructure of eggshell: a comparative study.

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.

Animals↗

Heat shock (45 degrees C) results in an increase of nuclear matrix protein mass in HeLa cells.

The nuclear matrix from HeLa cells heated at 45 degrees C was isolated to determine the effect of thermal shock on its composition and structure. The matrix from unheated cells contained about 10 per cent of total cell protein and was observed to be spherical particle with a diameter ranging from 3 to 5 microns with the major constituent polypeptides having molecular weights of 45, 47, 55, 57, 59 and 65 kilodaltons. The nuclear-matrix protein mass increased linearly with increasing exposure time at 45 degrees C with no observable change in its size or shape. The additional proteins were observed in general to have molecular weights greater than 45 kilodaltons, with marked increases in polypeptides of 28.5, 38.5, 60, 66, 75, 81, 88, 100 and 115 kilodaltons. An exponential relationship was observed between heat-induced cytotoxicity and the nuclear matrix protein mass increase. A 15 per cent increase in matrix protein mass was sustained prior to the onset of cytotoxicity, while a 35 per cent increase in matrix protein content was associated with a 63 per cent probability of cell killing. The results indicate that redistribution of cell protein or alterations in the mass or structure of the nuclear matrix may be involved in heat-induced cytotoxicity.

Cell Fractionation↗

Cross-linking in collagen by nonenzymatic glycation increases the matrix stiffness in rabbit achilles tendon.

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174-180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young's modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young's modulus of elasticity, and toughness (r values ranging from.61 to.94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from.22 to.84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.

Achilles Tendon↗