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Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction.

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 M guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 M NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.

Animals↗

In vitro chiral nematic ordering of chitin crystallites.

Microfibrillar fragments of purified crab and shrimp chitin were prepared by hydrolysis in 3 M HCl at its boiling point (104 degrees C). After removal of the acid by centrifugal washing and dialysis, an ultrasound treatment converts the residual product to a colloidal suspension stabilized by NH3+ charges. When dewatered to a critical concentration, spontaneous formation of a two-phase equilibrium system system occurs. The upper phase (lower concentration) is isotropic and the lower phase is anisotropic. The latter displays chiral nematic order and dries to a solid film which mimics the helicoid organization characteristic of the chitin microfibrils in the cuticle of arthropods.

Animals↗

Intermediate filament structure of alpha-keratin in baboon hair.

High-quality photographic recording of X-ray diffraction data from the hard alpha-keratins has mainly been limited to that from porcupine quill. Earlier diffraction patterns of hair have shown very few reflections. In the present study, extensive sets of high-quality data have been obtained using a synchrotron source and an image plate detector, revealing information about the internal structure, both axial and lateral. The diffraction patterns of hair from six different baboons have been examined. The meridional axial diffraction data reveal evidence of the superposition of three and possibly four separate lattices. Despite considerable overlapping, sufficient reflections unique to each lattice have been observed. These lattices comprise an infinite one of 46.76 nm, together with finite lattices of 19.67, 27.20 and 7.27 nm. These patterns reveal information regarding the fibrillar structure of the hair fibres, namely that it consists basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. There is also an indication of the presence of another set of cylinders of smaller diameter, possibly comprising the microfibrils of keratin.

Age Factors↗

Creep rupture of polypropylene sutures as a function of diameter, radiation dose and temperature.

The failure of polypropylene (PP) sutures was studied via creep rupture tests. Linear relationships were generated from plots of log time to break (tB) versus stress (sigma). At a constant sigma the results showed that tB was inversely proportional to the suture diameter (7-0, 6-0, 5-0 and 4-0) and to the radiation dose (0, 15, 20, 25 and 50 Mrad). Moreover, for the 6-0 suture size at a 15 Mrad dose, tB decreased with increasing temperature (26, 37, 44, 50 and 60 degrees C). For this creep rupture process the activation energy equalled 91.2 kJ/mol (21.8 kcal/mol). A mechanism for failure was presented which assumes that these sutures are bundles of oriented, semicrystalline microfibrils.

Dose-Response Relationship, Radiation↗

Ultrastructural investigation of circulating villous lymphoid cells: a tool in the differential diagnosis of splenic lymphoma with villous lymphocytes.

Ultrastructural examination of circulating lymphoid cells was performed in three cases of splenic lymphoma with circulating lymphocytes (SLVL) in order to define morphological features helpful to distinguish this lymphoma from hairy cell leukemia (HCL). The samples for ultrastructural investigation were obtained by Ficoll sedimentation from peripheral blood and routinely processed for electron microscopy. The ultrastructural features examined were: morphology of villi, morphology of nuclei, presence of nucleoli, distribution of heterochromatin, type of cytoplasmic organelles, presence of specific intracytoplasmic structures such as the ribosome-lamella complex, lysosome-like bodies and perinuclear microfibrils. Our results and a careful review of the literature seemed to confirm that SLVL has electron microscopic features typical enough to be relevant in the differential diagnosis with HCL.

Adult↗

Two very long periodicities in collagen.

A low-angle X-ray diffraction study of skin and breast-duct collagen, using synchrotron radiation, has disclosed an equatorial periodicity, common to all specimens, of about 350 nm. Tendons did not show any such periodicity, indicating that this phenomenon is restricted to two-dimensional distributions. In addition, shorter equatorial periodicities were also in evidence, which were in most instances different for different specimens, occurring in the range of 40 to 140 nm. Electron micrographs suggest the possibility that the first periodicity could be linked with blocks of the collagen molecule, which has a length of about 300 nm. The shorter periodicities give the tissue specific interfibrillar lateral packing of the individual collagen microfibrils.

Adult↗

Attachment of articular cartilage chondrocytes to the tissue form of type VI collagen.

Type VI collagen is composed of a short triple helix rich in RGD sequences with globular domains at each extremity of the helix. Disulfide-bonded tetramers of the monomeric molecule associate non-covalently to form networks of microfibrils in connective tissues, including cartilage. The disulfide-bonded tetramer can be extracted with 6 M guanidine HCl and purified without pepsin digestion and is referred to here as the tissue form of type VI collagen. Type VI collagen in mature articular cartilage appears to be concentrated pericellularly. We undertook a systematic investigation using solid phase assays to establish the nature of the attachment of bovine articular cartilage chondrocytes to the intact, tissue form of bovine type VI collagen. The tissue form of type VI collagen was extracted from bovine meniscus cartilage with 6 M guanidine HCl and purified by polyethylene glycol precipitation. When equal molar quantities were coated on microwells, the tissue form of type VI collagen attached more cells than the pepsin-digested form of the molecule that lacked the globular domains. The attachment to the intact, tissue form was dose-dependent and saturable and was not inhibited by heparin or type II collagen. A linear GRGDSP peptide failed to inhibit attachment of the chondrocytes to the intact, tissue or pepsin-digested forms of type VI collagen, but totally inhibited the interaction when the intact molecule was reduced and alkylated. In contrast, a cyclic C*GRGDSPC* peptide inhibited attachment to the tissue form of type VI collagen, but not to fibronectin. The attachment had a metal ion dependence that could be satisfied by MnCl2, slightly less by MgCl2, but not at all by CaCl2. A direct interaction between the tissue form of type VI collagen and a chondrocyte cell surface receptor or receptors is a structural feature of the pericellular matrix in cartilage.

Amino Acid Sequence↗

Histochemical characterization of collagen fibers in fibrous overgrowth (irritation fibroma) of the oral mucosa: effect of age and duration of lesion.

In a study of 43 specimens of oral mucosa irritation fibroma, polarization colours of collagen thin (0.8 microm) and thick (1.6-2.4 microm) fibers were recorded in formalin-fixed and paraffin-embedded sections stained with Picrosirius red. Examination of polarization colours of both the 'thin' and 'thick' collagen fibers revealed an increase of yellowish-orange and orange fibers as a function of age in patients over 30 years old (P < 0.05 only in thick fibers). When the polarization colours of thin and thick collagen fibers were recorded as a function of duration of the lesion, there was an increase in the number of yellowish-orange and orange fibers and a decrease in blue-green and green fibers in both thin (P < 0.1) and thick (P < 0.05) populations as the lesion matured. These findings show that increments in both the patient's age and the duration of the lesions increase the amount of collagen fibers which exhibit yellowish-orange and orange polarization colours indicating tighter packing and better alignment of the microfibrils, similar to normal mature collagen. These differences may have an effect on the timing of the surgical excision.

Journal Article↗

Amyloid elastosis: analysis of the role of amyloid P component.

We report the second case of amyloid elastosis. Our patient had an underlying primary systemic amyloidosis with lambda light chain paraproteinemia. Salient clinical features included a sclerodermatous facial appearance, cordlike thickening of superficial blood vessels, neck skin resembling that in pseudoxanthoma elasticum, livedo reticularis-like changes on the trunk, Raynaud's phenomenon, arterial and venous thromboses, and the nephrotic syndrome. Amyloid deposits were present in the dermis, around appendages, in blood vessel walls, and in a striking distribution surrounding individual elastic fibers, that appeared shortened and fragmented. Immunofluorescence, electron microscopic, and immunoultrastructural studies with antibodies to lambda light chain, localized the amyloid deposits to the region of the elastic fiber microfibrils, with which amyloid P component (AP) is invariably associated in normal tissues. Because AP binds amyloid fibrils, codistribution of amyloid deposits and AP in amyloid elastosis strongly supports the theory that elastic fiber-associated AP may act as a nidus for amyloid deposition.

Amyloidosis↗

Identification of elastic fibres in the peripheral nerve.

Traditional histological staining techniques, as well as elastin-specific antibodies and electron microscopy, have been used to assess the distribution of elastin within the peripheral nerve. The location of the elastin identified by the VerHoeff-VanGiesen or Weigert stains has been shown to coincide with the unambiguous identification of elastin by immunospecific stains and electron microscopy. Elastin is located in all three connective layers of the peripheral nerve. Thick elastic fibres, consisting of amorphous elastin protein and microfibrils, are located consistently in the perineurium and, to a lesser extent, in the epineurium. The endoneurium contains small collections of elastic fibres widely distributed between the axons. Compared with collagen, the overall content of elastin, however, is small, suggesting that the visco-elastic properties of peripheral nerve may be due primarily to collagen.

Animals↗

The palmar fascia after treatment by the continuous extension technique for Dupuytren's contracture.

After complete elongation using the continuous extension technique the palmar fascia of four patients with Dupuytren's contracture was examined by light and electron microscopy and compared with non-elongated samples from 20 patients at the same clinical stage of the disease. Nodules and cords were no longer clinically recognizable after extension. The tissue contained collagen fibrils of uniform diameter (about 50 nm), densely packed in fibres parallel to the stretching force. Fine filaments (presumably proteoglycans) formed a network which was intermingled with and periodically bound to the collagen fibrils. Fibroblasts and myofibroblasts with an high biosynthetic activity and oxytalan-like microfibrils were aligned along the collagen fibres. The results show that in Dupuytren's disease the contracted palmar fascia reacts to external forces with neoformation and reorientation of all tissue components by myofibroblasts.

Actin Cytoskeleton↗

Cellulose synthesis by Acetobacter xylinum. III. Matrix, primer and lipid requirements and heat stability of the cellulose-forming enzymes.

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone. The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycosylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.

Cell Wall↗

Dissociation of chitosomes by digitonin into 16 S subunits with chitin synthetase activity.

Digitonin exerts profound effects on chitosomes (microvesicular structures with chitin synthetase activity isolated from the fungus Mucor rouxii). At low concentrations, it stimulates chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity; at higher concentrations, it inhibits it. Digitonin also causes disintegration of the chitosome and the release of a homogeneous population of chitosome subunits with chitin synthetase activity. These chitosome subunits have a sedimentation coefficient of 16 S, compared to 105 S for whole chitosomes, as determined by centrifugation in sucrose density gradients, and measure 7--12 nm in diameter. After dissociation, chitin synthetase remains in a zymogenic state, and requires treatment with a protease for activation. No change in sedimentation coefficient of chitosome subunits was observed after proteolytic activation. The product synthesized by the chitosome subunits was characterized by X-ray diffractometry ad alpha-chitin and was by the criterion indistinfuishable from chitin made by preparations of undissociated chitosomes. However, in the electron microscope, the chitin microfibrils made from chitosome subunits were, in general, much shorter than those produced by undissociated chitosomes and often exhibited a needle-like appearance.

Centrifugation, Density Gradient↗

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells.

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.

Amino Acids↗

Matrix constituents of early developing bone examined by freeze fracture.

Specimens of aldehyde-fixed and glycerol-impregnated early developing bone matrix, obtained from rat calvaria, were examined by the freeze-fracture method. The developing bone matrix reveals collagen fibrils, numerous membranous structures and a granular background. The collagen fibrils, when viewed longitudinally, exhibit a substructure of thinner filaments (microfibrils) which appear to follow a twisted course along the fibril-axis. Some of the membranous structures are readily identified as osteoblast processes. Others, which are round, ovoid or irregular in shape, were found either with or without intramembrane particles (IMPs). It is concluded that the round or ovoid IMP-containing membranous structures correspond to matrix-vesicles. The nature of the IMP-free bodies, however, is uncertain. They may be artefacts or genuine matrix-vesicles deriving from unstable membrane domains which have a propensity for blebbing or budding off. Confirmation of the latter possibility might come from examination of directly frozen specimens.

Animals↗

Giant axonal neuropathy and leukodystrophy.

An 11-year-old Persian boy, born to consanguineous parents, manifested a progressive gait abnormality beginning at 5 years of age. A severe cerebellar disorder developed with associated dysfunction of the peripheral nervous system, but no sign of mental impairment. The sensory and motor nerve conduction velocities were greatly reduced, especially in the lower extremities. Cerebrospinal fluid protein was normal. Computed tomography and magnetic resonance imaging revealed leukoencephalopathy, especially in the cerebellum, but also in periventricular areas. The diagnosis of giant axonal neuropathy was established by biopsy of the sural nerve. The few previous histologic examinations have documented hyperplasia of the microfibrils which accumulate in the axons as well as in neurilemma, endothelial, and perineural cells. This is the first report of involvement of supraspinal portions of the central nervous system documented by postmortem examination after in vivo imaging methods corroborated the morphologic concomitants of the clinical symptoms.

Axons↗

Vitreous humor of chicken contains two fibrillar systems: an analysis of their structure.

An analysis of the structure of chicken vitreous humor after brief homogenization of the tissue was performed. Electron micrographs prepared after rotary shadowing with platinum showed the presence of two distinct fibrils. The collagen fibril was coated by glycosaminoglycan which could be removed by chondroitinase ABC digestion. In addition, individual molecules of tenascin were observed wrapped around some of the collagen fibrils. A second beaded fibril was present and several fine filaments were observed to extend from each bead. The beaded fibril is formed by the overlap of these filaments, and beaded fibrils were observed in either a "closed" or an "open" form dependent on whether all of the filaments are brought together to form the overlap. A schematic diagram is presented for the structure of the beaded fibril. The potential relationship of the beaded fibril to the zonular fibrils and the elastin microfibrils is briefly discussed.

Animals↗

Dermal collagen fibrils are hybrids of type I and type III collagen molecules.

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.

Adult↗