Insoluble Collagen Research Articles

The Impacts of Feeding Natursafe (an Immune Support Product) on Beef Quality

ObjectivesNaturSafe® (Diamond V, USA) is a Saccharomyces cerevisiae fermentation product developed as an animal feed supplement for the further manufacture of nutritionally balanced feeds for beef cattle. This immune support product (Association of American Feed Control Officials number 96.8, 73.046 and International Feed Name number 7–05–520, 8–08–034) has been specifically formulated to optimize beef cattle health and performance, antibiotic stewardship, and food safety. Research has shown that NaturSafe supports optimal rumen and liver health, overall health and immune function, consistency of feed intake, daily gain, feed conversion, and antibiotic effectiveness. As no research previously has assessed the impacts of this feed ingredient on the quality of meat, the objective of this research was to characterize the effects of feeding NaturSafe on meat quality characteristics in beef.Materials and MethodsCrossbred steers (N = 60, n = 12 per treatment; mean hot carcass weight = 421 kg), through an antibiotic free production system, were individually fed diets containing 12, 15, or 18 g/d of NaturSafe or a control diet without (–AB) antibiotics or a control with antibiotics (+AB; 330 mg monensin + 110 mg tylosin·steer–1·d–1) for 112 d. Strip loins were collected and aged for 13 or 29 d postmortem prior to fabrication. Steaks (m. Longissimus) were then evaluated for Warner-Bratzler shear force, pH, sarcoplasmic calcium concentration, troponin-T degradation, fatty acid profile, proximate composition, sarcomere length, total collagen and insoluble collagen. After each aging period, steaks were evaluated for lipid oxidation, and color characteristics (L*, a*, b*, discoloration percentage, and percentage surface oxymyoglobin, metmyoglobin and deoxymyoglobin), during and/or after a 7 d simulated retail display period. A subset of samples at various aging and retail display periods were analyzed for lactic acid bacteria (LAB), psychotrophic plate counts (PPC), and aerobic plate counts (APC). Animal was considered the experimental unit and hot carcass weight and marbling score were used as covariates in the analysis.ResultsTreatment had no effect on pH, sarcomere length, troponin-T degradation, fatty acid profile, proximate composition, total collagen, insoluble collagen, LAB, PPC, APC, lipid oxidation, oxymyoglobin percentage, or metmyoglobin percentage. Meat from cattle fed 18 g/d of NaturSafe was (1) equal to–AB controls and had higher shear force values compared to all other treatments (P < 0.01), (2) had higher (P < 0.05) sarcoplasmic calcium levels than +AB controls and cattle fed 12 g of NaturSafe/d, (3) was redder (higher a* values, P < 0.05) than all other treatments, and (4) was yellower (higher b* values, P < 0.01) than the 12 or 15 g dose and the–AB control. There were no differences among treatments fed NaturSafe for lightness (L*) at either aging time. There were no differences for meat from animals fed 12 or 15 g NatureSafe/d, except deoxymyoglobin percent and discoloration, which were both minimal. Discoloration values were low for all treatments (< 10%).ConclusionThese data indicate that feeding NaturSafe had few discernible effects on meat quality characteristics.

Cell-based Assay for Recruitment of DDR1 to Collagen-coated Beads.

The discoidin domain receptors, DDR1 and DDR2, are key signaling receptors for the extracellular matrix protein collagen. The interactions of cells with collagen are difficult to study because of the difficulty to obtain native collagen fibers for in vitro studies. Thus, in vitro studies often use acid-soluble collagens in the form of single triple helices, which are not representative of the densely packed insoluble collagen fibers found in tissues. In this protocol, we describe a method that allows stimulating DDR1 locally with collagen-coated beads. Latex beads are first coated with acid-soluble collagen, then added to cells expressing DDR1. Recruitment of DDR1 to the beads and collagen-induced DDR1 phosphorylation is visualized by immunofluorescence microscopy on a widefield microscope. In this method, densely packed collagen is presented to cells in an insoluble form. Bead coating is easy to perform, and this method thus presents a straightforward protocol with which to study local recruitment of collagen receptors to insoluble collagen.

Investigation of the Fatty Acid Profile of the M. rhomboideus Derived from Bos indicus Cattle

ObjectivesResearch has shown the m. Rhomboideus (Rho) from purebred Bos indicus (BI) to be unique in its proximate chemical composition, indicated by increased lipid deposition. Thus, potential for the depostion of mono- (MUFA) and poly- unsaturated (PUFA) fatty acids exists. The objective of this study was to assess the fatty acid composition of the intramuscular (IMF) and subcutaneous (SQF) depots in the Rho from BI crossed cattle.Materials and MethodsThree replications of 4 USDA Choice (Ch) and 4 USDA Select (Se) Rho muscles were selected from the right half of split carcasses (N = 24). Selection parameters were > 7.62-cm hump height, > 7.62-cm width, > 25.4-cm length, weight range: 2–4-kg. Muscles were removed from carcasses and vacuum packaged. After a 14-d aging period, Rho muscles were fabricated, 2.54-cm serially cut steaks (anterior to posterior), trimmed to 0.254-mm fat thickness. Steaks were assigned identification tags and designated for analysis. Rho steaks were used for proximate composition (n = 2), trained sensory analysis (n = 2), Warner-Bratzler shear force (WBS, n = 1), collagen content (n = 1), and fatty acid composition of IMF and SQF (n = 1). Steaks for trained sensory analysis were also used for color measurements and cook yield. Data were analyzed using a 2-sample t test. Sensory data were analyzed using a linear fit model with order as a random effect. All data analyzed using JMP v14.0.0. A predetermined significance level of P < 0.05 was used.ResultsTotal MUFA, PUFA and saturated fatty acid (SFA) percentages of IMF were not significant (P > 0.05) between Ch Rho muscles (43, 7.2, 47.1%, 0.89, respectively) and Se Rho muscles (43.0, 6.3, 50.7%, 0.86, respectively). Mean averages across both quality grades for total MUFA, PUFA, SFA, and MUFA:SFA (42.2, 6.8, 48.9%, 0.87, respectively) in Rho IMF were similar to reported FAC averages of Bos taurus (BT) longissimus dorsi IMF (47.8, 4.4, 47.8%, 1.0, respectively).Total MUFA percentage (49.1 vs. 45.4%) and MUFA:SFA ratio (1.1 vs. 0.9) were significantly higher (P < 0.001) in Ch SQF compared to Se SQF. However, total PUFA percentage (4.3 vs. 3.7%) and SFA (50.3 vs. 47.0%) were significantly higher (P < 0.02) in Se SQF compared to Ch SQF. Mean averages across both quality grade for total MUFA, PUFA, SFA, and MUFA:SFA (47.4, 4, 48.6%, 0.99, respectively) in SQF were similar to reported fatty acid averages of BT brisket SQF (56.8, 3, and 35.9%; 1.47, respectively).Protein content (19.4 vs. 18.6%) was higher (P < 0.02) for Ch than Se Rho muscles. L* value (50.2 vs. 47.9) was larger (P < 0.05) for Se. Ch Rho muscles contained greater amounts (P < 0.001) of total, insoluble, and soluble collagen (21.8, 21.5, 0.3 mg/g, respectively) compared to Se Rho muscles (13.8, 13.7, 0.1 mg/g, respectively). Ch Rho muscles were more tender (P < 0.001) as determined by WBS values (2.6 vs. 3.1 kg). Trained sensory analysis, pH, fat, moisture, a* and b* color values between quality grades were not different (P > 0.05).ConclusionDifferences were not seen for fatty acid composition between Ch and Se Rho IMF fat. However, higher percentages of total PUFA were found in both Ch and Se Rho IMF compared to reported longissimus dorsi IMF. Additionally, Ch Rho SQF contained higher percentages of total MUFA. However, Se Rho SQF contained higher percentages of total PUFA and SFA. Higher percentages of SFA were found in both Ch and Se SQF compared to reported brisket SQF SFA values.

Analysis of Physical, Mechanical and Thermal Degradation of Gelatin-Based Film–Exploring the Biopolymer for Plastic Advancement

Gelatin is an important natural biopolymer protein extracted from fibrous insoluble collagen and broadly used by various industries because of its stabilizing, and easy handling usage. Nowadays, scientists are interested to find out the alternative polymer of plastic, provide food safety as well having the properties of rapid degradation and environmental friendly. In this study, an investigation was carried out to figure out the property of gelatin as a bioplastic polymer. Gelatin based biofilms are produce in laboratory by solution casting method in Petri plates. The prepared biofilms characterized by tensile strength, break point elongation, swelling behavior, thermogravimetric analysis and degradation. Kinetic studies of pyrolysis of gelatin was investigated by using Friedman method and integral method apparent activation energy (E) were evaluated at constant heating rate. Food coating test were also employed to analysis the gelatin coating protection on food materials.

Analysis of Physical, Mechanical and Thermal Degradation of Gelatin-Based Film–Exploring the Biopolymer for Plastic Advancement

Gelatin is an important natural biopolymer protein extracted from fibrous insoluble collagen and broadly used by various industries because of its stabilizing, and easy handling usage. Nowadays, scientists are interested to find out the alternative polymer of plastic, provide food safety as well having the properties of rapid degradation and environmental friendly. In this study, an investigation was carried out to figure out the property of gelatin as a bioplastic polymer. Gelatin based biofilms are produce in laboratory by solution casting method in Petri plates. The prepared biofilms characterized by tensile strength, break point elongation, swelling behavior, thermogravimetric analysis and degradation. Kinetic studies of pyrolysis of gelatin was investigated by using Friedman method and integral method apparent activation energy (E) were evaluated at constant heating rate. Food coating test were also employed to analysis the gelatin coating protection on food materials.

An investigation on the correlation between the mechanical property change and the alterations in composition and microstructure of a porcine vascular tissue underwent trypsin-based decellularization treatment.

The nonlinear pseudoelastic behavior of a native/decellularized vascular tissue is closely related to the detailed composition and microstructure of the extracellular matrix and is important in maintaining the patency of a small-caliber vascular graft. A commonly used enzyme-detergent based decellularization protocol is effective in cell component removal but it also changes the microstructure and composition of the decellularized tissues. Previous studies provide limited information to correlate the mechanical property change with the alterations in composition and microstructure in a decellularization process. In this study, the correlations were studied by implementing a previously established fiber-progressive-engagement model to describe the nonlinear pseudoelastic behavior of a vascular tissue and to evaluate the effects of trypsin concentration and exposure duration on porcine coronary artery decellularization RESULTS: Results showed that tissue length and width increased and thickness and wet weight decreased with the exposure of trypsin. The effects of trypsin exposure times on the four mechanical parameters, i.e. initial strain, turning strain, initial modulus and stiffness modulus, in the longitudinal and circumferential directions were similar, but stronger in the circumferential direction. Major components of the extracellular matrix were vulnerable to the trypsin-based decellularization process. The decreases in initial and turning strain and the increase in initial modulus in circumferential direction were correlated with the significant decrease of collagen and glycosaminoglycans in the media layer. Although trypsin-based decellularization achieved cell component removal and preservation of ultimate tensile stress, the microstructure and composition changed with alterations in the pseudoelastic behavior of the porcine coronary artery. Taken together, the current observations suggested less waviness, early engagement, or re-alignment of insoluble collagen fibers in the media layer, which resulted in turning from anisotropic into isotropic uniaxial mechanical property of porcine vascular tissue. Selecting the proper trypsin concentration (< 0.03-0.5%) and duration (< 12 h) of trypsin exposure in combination with other methods will achieve optimal porcine coronary artery decellularization.

Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Regulatory T (Treg) cells offer new therapeutic options for controlling undesired systemic and local immune responses. The aim of the current study was to determine the impact of therapeutic Treg administration on systemic and cardiac inflammation and remodeling in coxsackievirus B3 (CVB3) -induced myocarditis. Therefore, syngeneic Treg cells were applied intravenously in CVB3-infected mice 3 d after infection. Compared with CVB3 + PBS mice, CVB3 + Treg mice exhibited lower left ventricular (LV) chemokine expression, accompanied by reduced cardiac presence of proinflammatory Ly6ChighCCR2highCx3Cr1low monocytes and higher retention of proinflammatory Ly6CmidCCR2highCx3Cr1low monocytes in the spleen. In addition, splenic myelopoiesis was reduced in CVB3 + Treg compared with CVB3 + PBS mice. Coculture of Treg cells with splenocytes isolated from mice 3 d post-CVB3 infection further demonstrated the ability of Treg cells to modulate monocyte differentiation in favor of the anti-inflammatory Ly6ClowCCR2lowCx3Cr1high subset. Treg-mediated immunomodulation was paralleled by lower collagen 1 protein expression and decreased levels of soluble and insoluble collagen in LV of CVB3 + Treg compared with CVB3 + PBS mice. In agreement with these findings, LV systolic and diastolic function was improved in CVB3 + Treg mice compared with CVB3 + PBS mice. In summary, adoptive Treg transfer in the inflammatory phase of viral-induced myocarditis protects the heart against inflammatory damage and fibrosis via modulation of monocyte subsets.-Pappritz, K., Savvatis, K., Miteva, K., Kerim, B., Dong, F., Fechner, H., Müller, I., Brandt, C., Lopez, B., González, A., Ravassa, S., Klingel, K., Diez, J., Reinke, P., Volk, H.-D., Van Linthout, S., Tschöpe, C. Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Acetic acid/1-ethyl-3-methylimidazolium acetate as a biphasic solvent system for altering the aggregation behavior of collagen molecules

A novel acetic acid/1-ethyl-3-methylimidazolium acetate (AA/[EMIM][Ac]) biphasic solvent system was adopted to alter the aggregation behavior of collagen molecules. Collagen aggregates (0.5 mg/mL) were investigated in the presence of AA/[EMIM][Ac] at various ratios (10:0 to 5:5). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the triple helical structural integrity was maintained integrity across the whole AA/[EMIM][Ac] ratio range. Ultra-sensitive differential scanning calorimetry measurements revealed that the transition temperature varied with the AA/[EMIM][Ac] ratio, indicating a relevant relationship between the aggregated sate of collagen molecules and thermal effects. The surrounding polarity of collagen molecules was examined by fluorescence spectroscopy of pyrene, and disaggregation behavior was enhanced with increasing [EMIM][Ac] ratio, as demonstrated by changes in the size of collagen aggregates determined by dynamic light scattering. Furthermore, the decrease in collagen aggregates was dependent on stronger interactions among collagen molecules. Intrinsic viscosity decreased from 11.526 to 2.607 g/dL, and the Huggins coefficient increased from 0.575 to 4.121 within the AA/[EMIM][Ac] ratio ranging from 10:0 to 5:5. Finally, atomic force microscopy visually confirmed the effect of [EMIM][Ac] on the aggregated state of collagen molecules visually. These findings suggest AA/[EMIM][Ac] could be an efficient solvent system for natural insoluble collagen.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Spectroscopic imaging: Nuclear magnetic resonance and Raman spectrometry for the detection of collagen cross-linking from giant squid mantle, fin, and tentacle tissues

ABSTRACTThe application of proton nuclear magnetic resonance (1H-NMR) imaging and Raman spectrometry is described for the detection and structural characterization of collagen cross-linking (pyridinoline) in soluble and insoluble collagen fractions obtained by cation-exchange separation of the mantle, fins, and tentacles of giant squid (Dosidicus gigas). The pyridinoline was detected using a fluorometric procedure only in the soluble collagen fractions. However, the pyridinoline was detected using high-performance liquid chromatography (HPLC) only in the insoluble collagen fraction from the tentacles. Meanwhile, in all samples, pyridinoline was detected by 1H-NMR and Raman spectrometry. The higher peak intensities in both methods were observed in the insoluble collagen fraction from the tentacles. The muscles showed a lower decrease in shear force after 30 min of cooking. 1H-NMR and Raman spectrometry provides an assay which is more sensitive than fluorescence and HPLC for pyridinoline detection in squid tissues, suggesting greater potential for the analysis of different types of tissue and collagen fractions.

Effect of Melatonin on the Renin-Angiotensin-Aldosterone System in l-NAME-Induced Hypertension.

The renin-angiotensin-aldosterone system (RAAS) is a dominant player in several cardiovascular pathologies. This study investigated whether alterations induced by l-NAME, (NLG)-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, and the protective effect of melatonin are associated with changes in the RAAS. Four groups of 3-month-old male Wistar rats (n = 10) were treated as follows for four weeks: untreated controls, rats treated with melatonin (10 mg/kg/day), rats treated with l-NAME (40 mg/kg/day), and rats treated with l-NAME + melatonin. l-NAME administration led to hypertension and left ventricular (LV) fibrosis in terms of enhancement of soluble, insoluble and total collagen concentration and content. Melatonin reduced systolic blood pressure enhancement and lowered the concentration and content of insoluble and total collagen in the LV. The serum concentration of angiotensin (Ang) 1–8 (Ang II) and its downstream metabolites were reduced in the l-NAME group and remained unaltered by melatonin. The serum aldosterone level and its ratio to Ang II (AA2-ratio) were increased in the l-NAME group without being modified by melatonin. We conclude that l-NAME-hypertension is associated with reduced level of Ang II and its downstream metabolites and increased aldosterone concentration and AA2-ratio. Melatonin exerts its protective effect in l-NAME-induced hypertension without affecting RAAS.

Evidence of glucuronidationof the glycation product LW-1: tentative structure and implications for the long-term complications of diabetes.

LW-1 is a collagen-linked blue fluorophore whose skin levels increase with age, diabetes and end-stage renal disease (ESRD), and correlate with the long-term progression of microvascular disease and indices of subclinical cardiovascular disease in type 1 diabetes. The chemical structure of LW-1 is still elusive, but earlier NMR analyses showed it has a lysine residue in an aromatic ring coupled to a sugar molecule reminiscent of advanced glycation end-products (AGEs). We hypothesized and demonstrate here that the unknown sugar is a N-linked glucuronic acid. LW-1 was extracted and highly purified from ~99g insoluble skin collagen obtained at autopsy from patients with diabetes/ESRD using multiple rounds of proteolytic digestion and purification by liquid chromatography (LC). Advanced NMR techniques (1H-NMR, 13C-NMR, 1H-13C HSQC, 1H-1H TOCSY, 1H-13C HMBC) together with LC-mass spectrometry (MS) revealed a loss of 176amu (atomic mass unit) unequivocally point to the presence of a glucuronic acid moiety in LW-1. To confirm this data, LW-1 was incubated with β-glycosidases (glucosidase, galactosidase, glucuronidase) and products were analyzed by LC-MS. Only glucuronidase could cleave the sugar from the parent molecule. These results establish LW-1 as a glucuronide, now named glucuronidine, and for the first time raise the possible existence of a "glucuronidation pathway of diabetic complications". Future research is needed to rigorously probe this concept and elucidate the molecular origin and biological source of a circulating glucuronidine aglycone.

Physicochemical changes of pepsin-solubilized and insoluble collagen in jumbo squid (Dosidicus gigas) muscle after cooking process

ABSTRACTCollagen is the major connective tissue (CT) protein and one of the main constituents of the jumbo squid (Dosidicus gigas). Therefore, physicochemical changes of pepsin-solubilized collagen (PSC) and insoluble collagen (IC) were studied after cooking (100°C/30 min) of muscle (mantle, fins, and arms). Different pyridinoline (Pyr) contents (the major cross-linking molecule in collagen fibers) were found in the fresh muscle of the three anatomical regions. After the cooking process, a decrease from 10 to 30% in the thermal resistance of collagen was observed, depending on the anatomical region and fraction evaluated. Furthermore, the electrophoretic profile, Fourier transform infrared (FTIR) spectroscopy, and the amino-acid profile revealed that structural changes occurred in the two different collagen fractions caused by the thermal process, and the changes were greater in the mantle. Under the conditions applied in this study, collagen fractions from the squid arms showed more stability during the cooking process due to the high cross-linking degree of their fibers.

Effects of Marbling Texture on Muscle Fiber and Collagen Characteristics

The objective of this study was to evaluate the effects of marbling texture on muscle fiber morphometrics, adipocyte cross-sectional area, perimysial thickness, collagen solubility, and thermal transition temperature of perimysial collagen of beef strip loin steaks from 3 marbling scores. Beef strip loins (n = 117) were selected from three marbling scores [Top Choice (Modest00 – Moderate100 marbling), Low Choice (Small marbling), and Select (Slight marbling)] to equally represent 3 different marbling texture groups: fine, medium, and coarse, via visual appraisal. There were no marbling texture × marbling score interactions for all traits evaluated. Marbling texture impacted (P &lt; 0.05) adipocyte cross-sectional area, where coarse steaks displayed larger adipocytes in comparison to fine marbled steaks, but medium marbled steaks were similar (P &gt; 0.05) to both coarse and fine marbled steaks for the same trait. However, marbling texture did not impact (P &gt; 0.05) perimysial thickness. Marbling texture did not affect collagen traits, as no differences (P &gt; 0.05) were found among marbling texture treatments for soluble collagen, insoluble collagen, and total collagen concentrations. Furthermore, all marbling texture groups (fine, medium, and coarse) were similar (P &gt; 0.05) for the peak thermal transition phase of the perimysial fraction of collagen. Marbling score affected adipocyte size, as Top Choice and Low Choice possessed larger (P &lt; 0.05) adipocytes than Select steaks. There were no differences (P &gt; 0.05) among marbling scores for fiber type; nor were differences found among marbling textures or marbling scores for fiber cross-sectional area. These results indicate that marbling texture does not contribute to differences in collagen characteristics or fiber cross-sectional area that may impact eating quality of beef strip loin steaks.

Effect of ractopamine on physicochemical characteristics and collagen solubility in finishing pigs

The objective of this study was to investigate physicochemical characteristics and collagen solubility of pigs fed with dietary ractopamine (RAC) during finishing period. Thirty of three-way crossbred pigs (Large White × Landrace × Duroc) with an initial bodyweight (BW) of 18.0 ± 0.5 kg were randomly assigned to one to three dietary RAC inclusions (0, 20, or 40 mg/kg) fed during finishing period (60-100 kg BW). Each group consisted of five gilts and five barrows. After 109 day of raising period, all pigs were slaughtered at the average weight of 100 kg. Longissimus dorsi muscle (LD) was taken from each carcass after overnight chill and analyzed for physical characteristics, chemical composition, and collagen solubility. The LD from pigs fed 40 mg/kg of RAC had lower a* value (p &lt; 0.01) than others. Pigs fed with 40 mg/kg of RAC had higher Warner-Bratzler shear force (p &lt; 0.05) than pigs fed without RAC. Pigs fed with dietary RAC had lower glycogen content than control (p &lt; 0.01). While other chemical compositions and collagen solubility of LD were not affected by dietary RAC (p &gt; 0.05). Gilts had larger muscle fibers (p &lt; 0.01), lower glycogen content (p &lt; 0.05), higher content of insoluble collagen (p &lt; 0.05), total collagen (p = 0.054), and collagen solubility (p = 0.054) than barrows.

AMS 14C Dating of Bones from Archaeological Sites in Mexico

ABSTRACTCollagen associated with bone samples is frequently used for radiocarbon (14C) dating of bones recovered from archaeological sites. However, submersion and exposure to moisture favors the degradation of collagen, which leads to difficulty in reliably dating bones from tropical, humid, or previously submerged archaeological sites. In this paper, we characterized the preservation state of a series of bones, through parameters such as %C, %N, C/N ratio, and collagen recovery. We performed 14C analyses of three collagen fractions obtained through the pretreatment steps (total, ultrafiltered, and insoluble collagen) in order to link the preservation state and the reproducibility of 14C values obtained from the three fractions. Collagen ultrafiltration resulted in a decrease of C/N ratio, although collagen yield was reduced. When two or three collagen fractions were obtained, ages were reproducible and consistent with expected values, according to archaeological or hydrogeological criteria. The pretreatment steps were monitored by infrared spectroscopy in order to analyze the collagen fractions at the molecular level. The presence of collagen in the total and insoluble fractions was confirmed. Since many of the Mexican samples had poor ultrafiltered collagen yield (&lt;3%) or nonexistent yield, our results show that if additional contextual information is carefully considered, the remnant collagen in the total and insoluble fraction can be dated, especially from sites where no other datable fraction exists.

LOX-related collagen crosslink changes act as an initiator of bone fragility in a ZDF rats model

Diabetes mellitus type 2 (DM2) results in bone abnormalities that manifest as increased bone fragility. Bone consists of two phases, the mineral phase and the matrix phase, and disorders in both are seen in DM2. However, the phase in which DM2 mediated bone fragility is initiated is still unknown. In this study, a male Zucker diabetic fatty (fa/fa) (ZDF) rat model was used to investigate the underlying mechanism initiating DM2 mediated bone fragility. The fracture surface morphology, pre- and post-yield bone mechanical behavior, insoluble collagen volume, lysyl oxidase family (LOX) enzyme levels and correlation analysis was performed to determine the relationship between insoluble collagen and post-yield behavior. Four weeks after the induction of diabetes, insoluble collagen was decreased in only the matrix phase in diabetic rats. Consistently, mechanical testing of the bone showed changes only in post-yield behavior. Diabetic rats also had decreased levels of enzymes involved in insoluble collagen formation (LOX and LOXL1 families). Correlation analysis demonstrated that insoluble collagen was a positive regressor of post-yield displacement (R = 0.894, P < 0.001) and post-yield energy (R = 0.918, P < 0.001). Together, these findings suggest that bone fragility in DM2 is initiated in the matrix phase and that the LOX family may play a critical part in the pathogenesis of DM2 mediated bone fragility.

Lysyl Oxidase-Like 1 Protein Deficiency Protects Mice from Adenoviral Transforming Growth Factor-β1-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The abnormal ECM deposition slowly overtakes normal lung tissue, disturbing gas exchange and leading to respiratory failure and death. ECM cross-linking and subsequent stiffening is thought to be a major contributor of disease progression and also promotes the activation of transforming growth factor (TGF)-β1, one of the main profibrotic growth factors. Lysyl oxidase-like (LOXL) 1 belongs to the cross-linking enzyme family and has been shown to be up-regulated in active fibrotic regions of bleomycin-treated mice and patients with IPF. We demonstrate in this study that LOXL1-deficient mice are protected from experimental lung fibrosis induced by overexpression of TGF-β1 using adenoviral (Ad) gene transfer (AdTGF-β1). The lack of LOXL1 prevented accumulation of insoluble cross-linked collagen in the lungs, and therefore limited lung stiffness after AdTGF-β1. In addition, we applied mechanical stretch to lung slices from LOXL1+/+ and LOXL1-/- mice treated with AdTGF-β1. Lung stiffness (Young's modulus) of LOXL1-/- lung slices was significantly lower compared with LOXL1+/+ lung slices. Moreover, the release of activated TGF-β1 after mechanical stretch was significantly lower in LOXL1-/- mice compared with LOXL1+/+ mice after AdTGF-β1. These data support the concept that cross-linking enzyme inhibition represents an interesting therapeutic target for drug development in IPF.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Proteolytic processing of lysyl oxidase–like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV.