Concentration and particle size distribution of polycyclic aromatic hydrocarbons formed by thermal cooking.

The concentration and particle size distribution of 19 major polycyclic aromatic hydrocarbons (PAHs) emitted by thermal cooking were investigated. Corn, trout, beef, prawns, and pork were selected for grilling. The PAHs in the oil mist emitted when the food was grilled were collected according to particle size range and analysed by GC/MS. Much higher concentrations of PAHs were detected in the oil mist emitted by grilled pork, trout, and beef samples, which were rich in fat. The main components of the cooking exhaust were 3- and 4-ring PAHs, regardless of food type. The particle size distribution showed that almost all the PAHs were concentrated in particles with diameters of <0.43 µm. For pork, the toxic equivalent of benzo[a]pyrene accounted for 50% of the PAHs in particles with diameters of <0.43 µm. From these results, we estimated that >90% of the PAHs would reach the alveolar region of the lungs.

Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin.

Tenomodulin (Tnmd, also called Tendin) is classified as a type II transmembrane glycoprotein and is highly expressed in developing as well as in mature tendons. Along with scleraxis (scx), Tnmd is a candidate marker gene for tenocytes. Its function is unknown, but it has been reported to have anti-angiogenic properties. Results in a knockout mouse model did not substantiate that claim. It has homology to chondromodulin-I. Single nucleotide polymorphisms of TNMD have been associated with obesity, macular degeneration, and Alzheimer's disease in patients. In the present study, three Tnmd isoforms with deduced molecular weights of 20.3 (isoform II), 25.4 (isoform III), and 37.1 (isoform I) kDa were proposed and verified by Western blot from cells with green fluorescent protein-linked, overexpressed constructs, tissue, and by qPCR of isoforms from human tissues and cultured cells. Overexpression of each Tnmd isoform followed by immunofluorescence imaging showed that isoforms I and II had perinuclear localization while isoform III was cytoplasmic. Results of qPCR demonstrated differential expression of each Tnmd isoform in patient's specimens taken from flexor carpi radialis, biceps brachii, and flexor digitorum profundus tendons. Knockdown of Tnmd increased the expression of both scleraxis (scx) and myostatin, indicating a potential negative feedback loop between Tnmd and its regulators. Knockdown of all Tnmd isoforms simultaneously also reduced tenocyte proliferation. I-TASSER protein three-dimensional conformation modeling predictions indicated each Tnmd isoform had different structures and potential functions: isoform 1, modeled as a cytosine methyltransferase; isoform 2, a SUMO-1-like SENP-1 protease; and isoform 3, an α-syntrophin, plextrin homology domain scaffolding protein. Further functional studies with each Tnmd isoform may help us to better understand regulation of tenocyte proliferation, tendon development, response to injury and strain, as well as mechanisms in tendinoses. These results may indicate novel therapeutic targets in specific tenomodulin isoforms as well as treatments for tendon diseases.

Mechanical loading stimulates ecto-ATPase activity in human tendon cells.

Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5'-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor-ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5'-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5-1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD3 and ENPP1, ENPP2). These data suggest that motion may release ATP from tendon cells in vivo, where ecto-ATPase may also be activated to hydrolyze ATP quickly. Ecto-ATPase may act as a co-modulator in ATP load-signal modulation by regulating the half-life of extracellular purine nucleotides. The extracellular ATP/ATPase system may be important for tendon homeostasis by protecting tendon cells from responding to excessive load signals and activating injurious pathways.

ATP modulates load-inducible IL-1beta, COX 2, and MMP-3 gene expression in human tendon cells.

Tendon cells receive mechanical signals from the load bearing matrices. The response to mechanical stimulation is crucial for tendon function. However, overloading tendon cells may deteriorate extracellular matrix integrity by activating intrinsic factors such as matrix metalloproteinases (MMPs) that trigger matrix destruction. We hypothesized that mechanical loading might induce interleukin-1beta (IL-1beta) in tendon cells, which can induce MMPs, and that extracellular ATP might inhibit the load-inducible gene expression. Human tendon cells isolated from flexor digitorum profundus tendons (FDPs) of four patients were made quiescent and treated with ATP (10 or 100 microM) for 5 min, then stretched equibiaxially (1 Hz, 3.5% elongation) for 2 h followed by an 18-h-rest period. Stretching induced IL-1beta, cyclooxygenase 2 (COX 2), and MMP-3 genes but not MMP-1. ATP reduced the load-inducible gene expression but had no effect alone. A medium change caused tendon cells to secrete ATP into the medium, as did exogenous UTP. The data demonstrate that mechanical loading induces ATP release in tendon cells and stimulates expression of IL-1beta, COX 2, and MMP-3. Load-induced endogenous IL-1beta may trigger matrix remodeling or a more destructive pathway(s) involving IL-1beta, COX 2, and MMP-3. Concomitant autocrine and paracrine release of ATP may serve as a negative feedback mechanism to limit activation of such an injurious pathway. Attenuation or failure of this negative feedback mechanism may result in the progression to tendinosis.

IL-1 beta induces COX2, MMP-1, -3 and -13, ADAMTS-4, IL-1 beta and IL-6 in human tendon cells.

Overuse injuries and trauma in tendon often involve acute or chronic pain and eventual matrix destruction. Anti-inflammatory drugs have been used as a treatment, however, the cellular and molecular mechanisms of the destructive processes in tendon are not clearly understood. It is thought that an inflammatory event may be involved as an initiating factor. Mediators of the inflammatory response include cytokines released from macrophages and monocytes. Interleukin-1 beta (IL-1 beta) is a candidate proinflammatory cytokine that is active in connective tissues such as bone and cartilage. We hypothesized that tendon cells would express receptors and respond to IL-1 beta in an initial "molecular inflammation" cascade, that is, connective tissue cell expression of cytokines that induce matrix destructive enzymes. This cascade results in expression of matrix metalloproteinases (MMPs) and aggrecanases that may lead to matrix destruction. Normal human tendon cells from six patients were isolated, grown to quiescence and treated with human recombinant IL-1 beta in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-1 beta (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-1, -3, -13 and aggrecanase-1 as well as IL-1 beta and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-1 beta-treated tendon cells released prostaglandin E(2) (PGE(2)) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE(2) was detectable at 10 pM IL-1 beta. IL-1 beta also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 beta. Post-injury or after some other inciting events, exogenous IL-1 beta released upon bleeding or as leakage of local capillaries may drive a proinflammatory response at the connective tissue cell level. The resulting induction of COX2, MMP-1 and -3 may underscore a potential for nonlymphocyte-mediated cytokine production of MMPs that causes matrix destruction and a loss of tendon biomechanical properties. Endogenous IL-1 beta might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.

IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P<0.001 for all comparisons). This is the first study documenting synergism of a signaling response to biomechanical and biochemical stimuli in human disc cells. IL-1beta treatment appeared to "sensitize" annulus cells to mechanical load. This increased responsiveness to mechanical load in the face of inflammatory cytokines may imply that the sensitivity of annulus cells to shear increases during inflammation and may affect initiation and progression of disc degeneration.

Insulin-like growth factor-I is expressed by avian flexor tendon cells.

Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48+/-0.05 ng/g tissue for the epitenon and 3.83+/-0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive form, most likely bound to insulin-like growth factor-binding proteins.

Shrinkage of perceived tonal duration produced by extra sounds: effects of spectral density, temporal position, and transition direction.

To investigate the mental mechanism that estimates the duration of sounds, the subjective duration of a tone was measured. In the first experiment a portion of the target sound was replaced with another sound. In the second and third experiments another sound either started or ended in the middle of the target sound. Ten or eleven undergraduates participated as listeners in each of the experiments. In the first experiment, effects of spectral spacing and those of temporal position of the replacing sound were tested. Compared with the intact case, the subjective duration of the replaced target shrunk, and the degree of shrinkage increased as the spectral spacing became wider. The temporal position of the replacing sound did not affect the degree of shrinkage. In the second experiment, effects produced by the start (ON) of the concurrent sound and those by the end (OFF) were compared. The ON case was more effective than the OFF case. In the third experiment, effects of the rise time and fall time of the concurrent sound were tested. A long rise and fall time reduced the difference between the ON and OFF cases. These results are discussed from two viewpoints, one assuming an interaction between the time markers, the other assuming continuous gate control in a neural-counting model for duration.

Gap junctions regulate responses of tendon cells ex vivo to mechanical loading.

Avian digital flexor tendons were used with a device to apply load ex vivo to study the effects on deoxyribonucleic acid and collagen synthesis when cell to cell communication is blocked. Flexor digitorum profundus tendons from the middle toe of 52-day-old White Leghorn chickens were excised and used as nonloaded controls, or clamped in the jaws of a displacement controlled tissue loading device and mechanically loaded for 3 days at a nominal 0.65% elongation at 1 Hz for 8 hours per day with 16 hours rest. Tendon samples were radiolabeled during the last 16 hours with 3H-thymidine to monitor deoxyribonucleic acid synthesis or with 3H-proline to radiolabel newly synthesized collagen. Cyclic loading of whole avian flexor tendons stimulated deoxyribonucleic acid and collagen synthesis, which could be blocked with octanol, a reversible gap junction blocker. Cells from human digital flexor tendon were used to populate a rectangular, three-dimensional, porous, polyester foam that could be deformed cyclically in vitro. Together, these results support the hypothesis that tendon cells must communicate to sustain growth and matrix expression and that an engineered three-dimensional construct can be used to study responses to mechanical load in vitro.

Post-stroke treatment with imidapril reduces learning deficits with less formation of brain oedema in a stroke-prone substrain of spontaneously hypertensive rats.

The present study was undertaken to examine the effects of the ACE (angiotensin converting enzyme) inhibitor imidapril, on the brain, when administered after the onset of stroke in a stroke-prone substrain of spontaneously hypertensive rats (SHRSP). Learning deficits and induced lesions in the brain as well as in the kidneys and heart were investigated in detail. SHRSP were divided into two groups with or without salt loading at the age of 4 weeks. The salt loading was performed for 7-9 weeks to increase the incidence of stroke. Within 24 h after the first observation of stroke, animals were subsequently treated with 5 mg/kg imidapril orally once a day or the vehicle for up to the age of 27 weeks. Imidapril attenuated progression of neurological abnormalities such as irritability, hyperkinesia and motor dysfunction, and increased survival rate. In three-panel runway testing, learning deficits did not develop significantly in the imidapril-treated group, and was comparable to that in the non-salt-loaded/non-stroke group. Imidapril reduced oedema formation in the cortex, hippocampus and striatum, and also suppressed lesion formation in the kidneys and heart. Imidapril thus suppressed progression of neurological deficits with loss of learning ability following onset of stroke, and also suppressed formation of oedema in the brain and decreased the number of lesions in other organs. Imidapril-induced reduction of cerebrovascular damage, which presumably occurs in the brain after stroke, may account for the inhibitory effects of imidapril on lesion formation and learning impairment.

Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells.

OBJECTIVE: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo. DESIGN: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro. METHODS: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1 h 45 min with the balance of time at rest to 6 h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1 Hz, 5% average elongation for 6 h, +/- PDGF-BB, IGF-I, TGF-beta 1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. CONCLUSIONS: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Acceptability for temporal modification of single vowel segments in isolated words.

Few perceptual studies of the temporal aspects of speech have investigated the influence of changes in segmental durations in terms of acceptability. Aiming to contribute to the assessment of rules for assigning segmental durations in speech synthesis, the current study measured the perceptual acceptability of changes in the segmental duration of vowels as a function of the segment attributes or context, such as base duration, temporal position in a word, vowel quality, and voicing of the following segment. Seven listeners estimated the acceptability of word stimuli in which one of the vowels was subjected to a temporal modification from -50 ms (for shortening) to +50 ms (for lengthening) in 5-ms steps. The temporal modification was applied to vowel segments in 70 word contexts; their durations ranged from 35-145 ms, the mora position in the word was first or third, the vowel quality was /a/ or /i/, and the following segment was a voiced or an unvoiced consonant. The experimental results showed that the listeners' acceptable range of durational modification was narrower for vowels in the first moraic position in the word than for those in the third moraic position. The acceptable range was also narrower for the vowel /a/ than for the vowel /i/, and similarly narrower for vowels followed by unvoiced consonants than for those followed by voiced consonants. The vowel that fell into the least vulnerable class (the third /i/, followed by a voiced consonant) required 140% of the modification of that which fell into the most vulnerable class (the first /a/, followed by an unvoiced consonant) to yield the same acceptability decrement. In contrast, the effect of the original vowel duration on the acceptability of temporal modifications was not significant despite its wide variation (35-145 ms).

Acceptability for temporal modification of consecutive segments in isolated words.

Perceptual sensitivity to temporal modification in two consecutive speech segments was measured in word contexts to explore the following two questions: (1) whether there is an interaction between multiple segmental durations, and (2) what aspect of the stimulus context determines the perceptually salient temporal markers? Experiment 1 obtained acceptability ratings for words with temporal modifications. The results showed that the compensatory change in duration of a vowel (V) and its adjacent consonant (C) is not perceptually so salient as expected for the simultaneous modifications in the two segments. This finding suggests the presence of a time perception range wider than a single segment (V or C). The results of experiment 1 also showed that rating scores for compensatory modification between V and C do not depend on the temporal order of modified pairs (VC or CV), but rather on the loudness difference between V and C; the acceptability decreased when the loudness difference between V and C became high. This suggests that perceptually salient markers locate around major jumps in loudness. The second finding, the dependence on the loudness jump, was replicated in experiment 2, which utilized a detection task for temporal modifications on nonspeech stimuli modeling the time-loudness features of the speech stimuli. Experiment 3 further investigated the influence of the temporal order of V and C by utilizing the detection task for the speech stimuli instead of the acceptability ratings.

PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro.

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp60src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.

Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals.

Cells from diverse tissues detect mechanical load signals by similar mechanisms but respond differently. The diversity of responses reflects the genotype of the cell and the mechanical demands of the resident tissue. We hypothesize that cells maintain a basal equilibrium stress state that is a function of the number and quality of focal adhesions, the polymerization state of the cytoskeleton, and the amount of extrinsic, applied mechanical deformation. A load stimulus detected by a mechano-electrochemical sensory system, including mechanically sensitive ion channels, integrin-cytoskeleton machinery, and (or) a load-conformation sensitive receptor or nonreceptor tyrosine kinase, may activate G proteins, induce second messengers, and activate an RPTK or JAK/STAT kinase cascade to elicit a response. We propose the terms autobaric to describe a self-loading process, whereby a cell increases its stress state by contracting and applying a mechanical load to itself, and parabaric, whereby a cell applies a load to an adjacent cell by direct contact or through the matrix. We predict that the setpoint for maintaining this basal stress state is affected by continuity of incoming mechanical signals as deformations that activate signalling pathways. A displacement of the cytoskeletal machinery may result in a conformational change in a kinase that results in autophosphorylation and cascade initiation. pp60Src is such a kinase and is part of a mechanosensory protein complex linking integrins with the cytoskeleton. Cyclic mechanical load induces rapid Src phosphorylation. Regulation of the extent of kinase activation in the pathway(s) may be controlled by modulators such as G proteins, kinase phosphorylation and activation, and kinase inhibitors or phosphatases. Intervention at the point of ras-raf interaction may be particularly important as a restriction point.

Fibronectin in the tendon-synovial complex: quantitation in vivo and in vitro by ELISA and relative mRNA levels by polymerase chain reaction and northern blot.

An enzyme-linked immunosorbent assay was used to quantitate fibronectin (Fn) levels in the outer synovia (epitenon) and internal fibrous portion (endotenon) of chicken flexor tendon and sheath. Primary cell cultures from these tissues and their secretions also were assayed for Fn levels. The polymerase chain reaction (PCR) was used to determine relative steady-state levels of Fn mRNA in primary cultures of synovial and internal fibroblasts from chicken tendon, and Northern blot analysis was performed to verify relative levels of the Fn message. The epitenon contained 3.8-fold more Fn than did the endotenon, and the sheath synovium contained 21-fold more Fn than did the internal fibrous portion of sheath. Cells cultured from the epitenon produced 9.3 and 13-fold more cell-associated and secreted Fn, respectively, than did cultured endotenon fibroblasts. Sheath synovial cells produced 17 and 3.2-fold more cell-associated and secreted Fn, respectively, than did sheath internal fibroblasts. Levels of Fn mRNA, as measured by PCR and Northern blot, were 1.6 and 1.8-fold greater, respectively, in tendon synovial cells compared with tendon internal fibroblasts. The biologic reason for increased Fn in tendon synovium is not known. We theorize that Fn may stabilize tendon synovium to shear stress and may play a role in the modulation of synovial rheology in the normal tendon. In the injured tendon, Fn may be involved in the organization of collagen deposition or may act through association with growth factors to aid healing.

Tendon collagens: extracellular matrix composition in shear stress and tensile components of flexor tendons.

Outer synovial tissues were separated from the remainder of avian flexor tendon and the collagens characterized biochemically and compared with those of the internal portion of tendon and sheath. The collagen content of tendon synovium was 23%, whereas that of tendon and sheath were 78% and 73%, respectively, based on dry weight. Four genetic types of collagen were found in the pepsin solubilized matrices: in the synovium, types I (78%) and III (19%) predominated; types V and possibly VI were present as minor collagens. Purified synovial type V collagen was a heterotrimer, with chain composition [alpha 1(V)]2 alpha 2(V). In contrast, the internal portion of tendon and sheath were comprised of only type I collagen. There was a large amount (41%) of ethanol extractable, noncollagenous material present in synovium, a part of which was proteoglycans. In addition, collagen cross-links of these tissues were quantified: the internal tendon had an abundant concentration of pyridinoline; synovium exhibited high amounts of labile, reducible cross-links, particularly dihydroxylysinonorleucine. In the case of sheath, lysine aldehyde-derived cross-links appeared to be predominant. These results indicate that each tissue has its own collagen type distribution as well as cross-linking pattern reflecting their maturational and functional differences.

Amino-terminal location of pyridinoline in dentin collagen.

Cross-linking is believed to be one of the major factors that characterize the calcifiability of dentin and bone collagens. Dehydro-dihydroxylysinonorleucine and pyridinoline which constitute the principal cross-links of dentin collagen have so far been located only in the carboxy terminal telopeptide of the molecules [alpha 1(I)-chain 87 x alpha 1(I)-chain 16C]. This situation suggested that the amino terminal telopeptide portion might be "open" without intermolecular cross-linking in hard tissue collagen fibrils. However, the present study provided evidence that pyridinoline is also located in amino-terminal telopeptides (alpha 1-chain 9N or alpha 2-chain 5N) and alpha 1-chain 930. Bovine dentin collagen was digested with trypsin followed by heating at 60 degrees C before and after the digestion. This method gave complete trypsin peptides of dentin collagen. Fluorescent pyridinoline peptides with a smaller molecular size were isolated by Sephadex G-50 superfine, DEAE-cellulose and reverse-phase HPLC. Automatic Edman analysis of several isolated peptides revealed the five-residue sequence, Gly-Ile-X-Gly-His-Arg, the only assignment of which was alpha 1-chain 928-933. The above evidence together with the amino acid compositions of the peptides led to the conclusion that pyridinoline is located not only in the carboxy-terminal but also in the amino-terminal telopeptide in dentin collagen.

Collagen in the spicule organic matrix of the gorgonian Leptogorgia virgulata.

Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. The insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed.

Paracoccus kocurii sp. nov., a tetramethylammonium-assimilating bacterium.

A new species of tetramethylammonium-assimilating bacteria was isolated from an activated sludge which was used for the treatment of tetramethylammonium hydroxide contained in the wastewater from semiconductor manufacturing processes. Cells of the bacteria were gram-negative, nonmotile, short rods (0.5 to 0.8 micron by 0.7 to 1.1 microns). The major respiratory quinone component of the bacteria was Q-10. The G + C content was 71 mol%. Isolates are mesophilic and assimilate methylated amines such as tetramethylammonium, trimethylamine, dimethylamine, and methylamine under neutral conditions. The isolates resemble Paracoccus species with respect to morphology but were distinguishable from the known species of the genus. We propose Paracoccus kocurii sp. nov. The type strain is strain B (= JCM 7684).