Low angle X-ray diffraction studies on stained rat tail tendons

Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0--17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0--20.0 nm in collagen fibrils under some disruptive or developmental conditions.     

Arrangement of microfibrils in collagen fibrils of tendons in the rat tail

Summary The microfibrillar arrangement in collagen fibrils of tendons in the tail of the rat was examined by electron microscopy and X-ray diffraction. Fresh and air-dried collagen fibers were examined in unstretched and stretched conditions. The results demonstrate that the microfibrils have a course parallel to the longitudinal axis of the collagen fibrils. The influence of stretching and hydration of the samples on the orientation of fibrils and microfibrils is also assessed.     

Structure and function of tuna tail tendons

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.     

Changes in the cross-linking of collagen from rat tail tendons due to diabetes

The acid solubility of Type I collagen from rat tail tendons decreases due to diabetes. This finding has been taken as evidence that collagen from diabetics may be more cross-linked than normal. We compared CNBr peptide maps prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for [3H] NaBH4-reduced tail tendons from streptozotocin-diabetic rats with maps from age-matched control rats. At least through 30 weeks of diabetes, the distribution of mass of both cross-linked and uncross-linked CNBr peptides was identical in diabetic and control tendons. Therefore, the number of cross-linked peptides did not increase due to diabetes. We analyzed the 3H-cross-linking compounds present on the CNBr peptides and found that the 3H content of peptides cross-linked in control tendons through the bivalent, reduced cross-links hydroxylysinonorleucine and lysinonorleucine was diminished on corresponding peptides from diabetic tendons as a function of duration of diabetes. The cross-linked peptides, however, persisted. Therefore, we conclude that a larger fraction of these bivalent cross-links is found in an unknown, non-reducible form in tendons from diabetic compared with control rats. This resembles a phenomenon normally associated with maturation and/or aging where the non-reducible form of the cross-links is acid-stable. An increase in the fraction of the cross-links that is non-reducible and acid-stable would explain, at least in part, the decrease in acid solubility of the collagen. Non-enzymatic glycation (NEG) was not very specific, since most CNBr peptides bound some glucose. However, peptides from the alpha 2-chain seemed to be preferential targets for NEG. While NEG clearly increased due to diabetes, we found no evidence that increased NEG led to an increased number of cross-links in tail tendon collagen from streptozotocin diabetic rats.     

Regional adaptations in three rat tendons

Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

Evidence for basement membranes in rat tail tendon sheaths

The presence of anti-laminin antibodies and a basement membrane-like thin electrondense lamina has been demonstrated in the peritendineum of the rat tail tendon by indirect immunofluorescence and electron microscopy.     

Changes in solubility, non-enzymatic glycation, and fluorescence of collagen in tail tendons from diabetic rats

The increase in acid-insoluble collagen (AIC) from tail tendons of streptozotocin-diabetic rats was measured and compared with that for control rats. AIC increased from 10% initially to 75% after 12 weeks of diabetes. It then increased slowly to 85% after 45 weeks. AIC for control rats was constant for the first 12 weeks and then increased slowly to 40% after 45 weeks. These data are consistent with an increase in the number of acid-stable cross-links in the collagen due to diabetes. The quantity of collagen solubilized by pepsin at 4 degrees C was unaltered due to diabetes, strong evidence that formation of diabetes-induced cross-links between helical regions of collagen molecules cannot explain the increase in AIC observed. Non-enzymatic glycation (NEG) increased linearly over 45 weeks, but the rate of NEG was much slower than the rate of increase in AIC observed for diabetics. The level of NEG for diabetics was about three times that for controls at a given time, but there was still less than 1 mol of glucose detected/mol of collagen at near maximum acid insolubility. Fluorescence associated with tail tendons was measured to test the hypothesis that fluorescent cross-links form as a consequence of NEG and result in decreased collagen solubility. Fluorescence (lambda ex 370; lambda em 430) increased slowly with age but was similar for control and diabetic tendons of the same age. Fluorescence was not increased in AIC compared with acid-soluble collagen derived from a given tendon sample. NEG of collagen reached near-diabetic levels in non-diabetic rats whose growth was inhibited by restricted feeding, but there was no associated increase in AIC. These data suggest that NEG and the subsequent formation of fluorescent cross-links do not contribute significantly to the rapid increase in AIC in the streptozotocin-rat model of diabetes.     

Distribution of hyaluronan in human endometrium across the menstrual cycle

Hyaluronan is a molecule with many known roles in cellular physiology and is often associated with tissue remodeling. The human endometrium undergoes dramatic remodeling during the course of the normal menstrual cycle but its regulation is not well understood. This study examined the levels of deposition of hyaluronan in human cycling endometrium by histochemical localization, using a highly specific hyaluronan-binding peptide. This was facilitated by avoiding conventional formalin fixation, which results in serious loss of the water-soluble hyaluronan. Peaks of hyaluronan deposition were observed in the stromal compartment during the mid-proliferative (days 5-10) and the mid-secretory phase (days 19-23) of the cycle. In physiological terms, the first phase corresponds to the time of rapid cellular proliferation of undifferentiated cells, whereas the second phase coincides with the time when the implantation of the conceptus would be initiated in a fertile cycle. By the time menstruation starts, very little hyaluronan remains in the stroma. In contrast with the stromal staining, hyaluronan deposition around blood vessels was constant throughout the cycle. The dramatic changes in hyaluronan deposition and their correlation with the cyclic growth and remodeling in the human endometrium suggest a major role for hyaluronan in the physiology of this tissue.     

Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.     

Angiotensin converting enzyme inhibition blocks interstitial hyaluronan dissipation in the neonatal rat kidney via hyaluronan synthase 2 and hyaluronidase 1

A functional renin-angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6-8 days old control rats cortical HA content was high and reduced by 93% on days 10-21, reaching adult low levels. Medullary HA content was high on days 6-8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.     

Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.     

Distribution of albumin variants in Indians and non-Indians of Mexico

The distribution of albumin variants amongst several Mexican Indian and non-Indian (Mestizo) groups was studied. Of the former, a total of 1606 individuals belonging to 11 different tribes were examined with an overall frequency of 1.5% of Albumin Mexico fairly uniformly distributed in all four main linguistic groups. The 2548 Mestizos studied belong in six groups, two from Mexico City and four from elsewhere in the country. The first of the Mexico City groups consisted of 1313 individuals randomly chosen from the outpatient clinic of the Instituto Nacional de la Nutrición, while the rest included healthy individuals. The overall frequency was 1.9%, also fairly evenly distributed, with no difference between the hospital population and the others. No anomalous albumin, other than Albumin Mexico was encountered in the whole study. It is concluded that the similarity between Indians and Mestizos is due to the high Indian component of the latter and that the presence of albumin Mexico is a good anthropological marker for this region of the world.     

Simple method for bleeding the unanaesthetized rat by tail venipuncture

A technique is described for the intermittent collection of blood from the rat tail. By using commonly available equipment, blood samples can easily be obtained from rats without the need for anaesthesia. The development of this technique makes the rat more readily available as an animal model for repeated withdrawals of small blood samples for pharmacokinetic or bioavailability evaluations.     

Distribution of albumin in normal and regenerating livers of mice

Summary The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with ³H-valin and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules.In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G₁- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and ³H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.Supported by a grant from the Robert-Bosch-Foundation, Stuttgart, Federal Republic of Germany     

Age-dependent changes in the mechanical properties of tail tendons in TGF-beta inducible early gene-1 knockout mice.

The purpose of this study is to investigate age-dependent changes in the architecture and mechanical properties of tendon in TGF-beta inducible early gene-1 (TIEG) knockout mice. Wild-type and TIEG knockout mice, aged 1, 2, and 15 mo, were used. The mechanical properties of tail tendons isolated from these mice were determined using uniaxial tensile ramp (0.05 mm/s) and relaxation (5 mm/s) tests, with a strain of 10%. Mechanical parameters (Young's modulus from the ramp test; fast and static stresses from the relaxation test) were measured and recorded. The structure of the tail tendon fascicle was characterized by transmission electron microscopy. The results of the mechanical testing revealed no significant difference between the knockout and wild-type groups at 1 or 15 mo of age. However, the fascicles of the knockout mice at 3 mo of age exhibited decreased fast and static stresses compared with those of the wild-type mice. Electron microscopy revealed an increase in fibril size in the knockout mouse tendons relative to wild-type controls at 1 and 3 mo of age. These data indicate an important role for TIEG in tendon microarchitecture and strength in adult mice.