Non-fibrillar components of amyloid deposits mediate the self-association and tangling of amyloid fibrils

Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, beta-sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.     

Identification of components of the extracellular matrix in trophoblastic cell columns of human placenta]

The distribution of five components of the extracellular matrix was studied in human placenta (9-12 and 39-40 weeks of gestation) by an indirect immunofluorescence method with polyclonal monospecific antibodies. In trophoblastic cell columns fibronectin, collagen types IV and V formed homogeneous deposits, whereas collagen types I and II comprised small conglomerates and scanty, discrete granules. The origin of these macromolecules was discussed.     

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats.     

Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae)

Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries.     

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.     

The effect of loading rate on the development of early damage in articular cartilage

Experimental reports suggest that cartilage damage depends on strain magnitude. Additionally, because of its poro-viscoelastic nature, strain magnitude in cartilage can depend on strain rate. The present study explores whether cartilage damage may develop dependent on strain rate, even when the presented damage numerical model is strain-dependent but not strain-rate-dependent. So far no experiments have been distinguished whether rate-dependent cartilage damage occurs in the collagen or in the non-fibrillar network. Thus, this research presents a finite element analysis model where, among others, collagen and non-fibrillar matrix are incorporated as well as a strain-dependent damage mechanism for these components. Collagen and non-fibrillar matrix stiffness decrease when a given strain is reached until complete failure upon reaching a maximum strain. With such model, indentation experiments at increasing strain rates were simulated on cartilage plugs and damage development was monitored over time. Collagen damage increased with increasing strain rate from 21 to 42 %. In contrast, damage in the non-fibrillar matrix decreased with increasing strain rates from 72 to 34 %. Damage started to develop at a depth of approximately 20 % of the sample height, and this was more pronounced for the slow and modest loading rates. However, the most severe damage at the end of the compression step occurred at the surface for the plugs subjected to 120 mm/min strain rate. In conclusion, the present study confirms that the location and magnitude of damage in cartilage may be strongly dependent on strain rate, even when damage occurs solely through a strain-dependent damage mechanism.     

Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry

Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A-D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Furthermore, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.     

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development

Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.     

Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by snake venom metalloproteinases

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels.     

Anti-basement membrane glomerulopathy in experimental trypanosomiasis

The nature of kidney lesions in BD IX rats infected with Trypanosoma brucei was investigated. Proteinuria developed and increased up to 236 +/- 35 mg/24 hr at 7 wk after the infection. Antibodies were found to be deposited along the glomerular basement membrane (GBM) predominantly in a linear fashion, which changed to a more granular pattern 7 wk after the infection. At this stage of the disease, electron-dense deposits were found subendothelially along the GBM. In the sera and kidney eluates of diseased rats, anti-GBM antibodies were present. Enzyme-linked immunosorbent assay (ELISA) studies showed antibodies which reacted with GBM components laminin and type IV collagen and not with fibronectin. The antibody specificity was confirmed by using competitive and cross-absorption ELISA techniques, as well as immunoblotting. With the use of indirect immunofluorescence, no common antigenic sites were found on trypanosomes and GBM components. The observed linear immunofluorescence pattern seems to be caused by glomerular binding of antibodies directed against laminin and type IV collagen, which are known to be able to induce renal disease. Subendothelial complex formation in later stages of the disease might result from a molecular rearrangement of GBM components after in situ binding of the antibodies. The formation of auto-antibodies directed against laminin and type IV collagen is probably caused by restricted polyclonal B cell stimulation, a well known feature of trypanosomiasis.     

Stress distribution and consolidation in cartilage constituents is influenced by cyclic loading and osteoarthritic degeneration

The understanding of load support mechanisms in cartilage has evolved with computational models that better mimic the tissue ultrastructure. Fibril-reinforced poroelastic models can reproduce cartilage behaviour in a variety of test conditions and can be used to model tissue anisotropy as well as assess stress and pressure partitioning to the tissue constituents. The goal of this study was to examine the stress distribution in the fibrillar and non-fibrillar solid phase and pressure in the fluid phase of cartilage in axisymmetric models of a healthy and osteoarthritic hip joint. Material properties, based on values from the literature, were assigned to the fibrillar and poroelastic components of cartilage and cancellous and subchondral compact bone regions. A cyclic load representing walking was applied for 25 cycles. Contact stresses in the fibrillar and non-fibrillar solid phase supported less than 1% of the contact force and increased only minimally with load cycles. Simulated proteoglycan depletion increased stresses in the radial and tangential collagen fibrils, whereas fibrillation of the tangential fibrils resulted in increased compressive stress in the non-fibrillar component and tensile stress in the radial fibrils. However neither had an effect on fluid pressure. Subchondral sclerosis was found to have the largest effect, resulting in increased fluid pressure, non-fibrillar compressive stress, tangential fibril stress and greater cartilage consolidation. Subchondral bone stiffening may play an important role in the degenerative cascade and may adversely affect tissue repair and regeneration treatments.     

Dipeptidyl peptidase (DPP) IV in rat organs

Summary Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Further-more, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.Dedicated to Professor Z. Lojda on the occasion of his 60th birthdayTo whom offprint requests should be sentSupported by the Deutsche Forschungsgemeinschaft (Re 523/2-1), Hermann and Lilly Schilling-Stiftung and Maria Sonnenfeld-Stiftung     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

A new twist in the collagen story--the type VI segmented supercoil

Collagen occurs in two major forms: fibrillar and non-fibrillar. Non-fibrillar collagens are structurally more variable and relatively ill-understood. In this work we analysed the amino acid sequence of type VI collagen, a non-fibrillar collagen that forms antiparallel dimers. A sequence motif was discovered that gives rise to systematic molecular coiling. There is a common periodicity ( approximately 23 or 2 x 23 residues) in the charged amino acids, in the prolines and in the discontinuities in the Gly-X-Y triplets. In addition, there is a different periodicity ( approximately 21 amino acids) in the apolar groups. The two repeats mean that the only way to simultaneously maximize both the hydrophobic and polar interactions during dimer formation is with the molecules antiparallel, overlapped by 75 nm as observed, and supercoiled. The alternating proline-rich and charge-rich patches, often together with discontinuities in the Gly-X-Y sequences, coincide with each half-turn of the supercoil, thus breaking it into segments. We have termed this structure the collagen segmented supercoil. The segmented supercoil and variants may be common aggregation motifs for the non-fibrillar collagens.     

Fermentation products, amino acid utilization, maintenance energies and growth yields for the fibrillar Streptococcus salivarius HB and a non-fibrillar mutant HB-B grown in continuous culture under glucose limitation

The fibrillar strain Streptococcus salivarius HB and a non-fibrillar mutant, strain HB-B, were grown in a defined medium under glucose limitation in a chemostat. Fermentation balances were produced for both strains in batch culture and at growth rates between 0.1/h and 1.1/h. In batch culture both strains fermented glucose to lactate, but in continuous culture glucose was fermented to formate, acetate and ethanol with increasing amounts of lactate as the growth rate was increased. Lactate never became the major fermentation product even at the highest growth rate. Amino acid analysis showed that only lysine was more than 50% utilized, while proline and tyrosine showed net production. The non-fibrillar strain HB-B showed, in general, a reduced utilization of amino acids compared with the fibrillar strain HB. Calculated growth yields and maintenance energies for the two strains showed that there was a reduction in the true growth yield and the maintenance energy coefficient of the non-fibrillar strain HB-B when compared with the fibrillar strain HB. The increase in the maintenance energy of the fibrillar strain HB (1.382 mmol/g/h) when compared with the non-fibrillar strain HB-B (0.546 mmol/g/h) of 153% is proposed to be the energy required for the maintenance of the fibrillar surface of the cell.     

Fermentation products, amino acid utilization, maintenance energies and growth yields for the fibrillar Streptococcus salivarius HB and a non-fibrillar mutant HB-B grown in continuous culture under glucose limitation

The fibrillar strain Streptococcus salivarius HB and a non-fibrillar mutant, strain HB-B, were grown in a defined medium under glucose limitation in a chemostat. Fermentation balances were produced for both strains in batch culture and at growth rates between 0.1/h and 1.1/h. In batch culture both strains fermented glucose to lactate, but in continuous culture glucose was fermented to formate, acetate and ethanol with increasing amounts of lactate as the growth rate was increased. Lactate never became the major fermentation product even at the highest growth rate. Amino acid analysis showed that only lysine was more than 50% utilized, while proline and tyrosine showed net production. The non-fibrillar strain HB-B showed, in general, a reduced utilization of amino acids compared with the fibrillar strain HB. Calculated growth yields and maintenance energies for the two strains showed that there was a reduction in the true growth yield and the maintenance energy coefficient of the non-fibrillar strain HB-B when compared with the fibrillar strain HB. The increase in the maintenance energy of the fibrillar strain HB (1.382 mmol/g/h) when compared with the non-fibrillar strain HB-B (0.546 mmol/g/h) of 153% is proposed to be the energy required for the maintenance of the fibrillar surface of the cell.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II-III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderant reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subunits I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.