Attila Bardosi

Myo-, neuro-, gastrointestinal encephalopathy (MNGIE syndrome) due to partial deficiency of cytochrome-c-oxidase

SummaryA 42-year-old woman had a 10-year history of external ophthalmoplegia, malabsorption resulting in chronic malnutrition, muscle atrophy and polyneuropathy. Computer tomography revealed hypodensity of her cerebral white matter. A metabolic disturbance consisted of lactic acidosis after moderate glucose loads with increased excretion of hydroxybutyric and fumaric acids. Post-mortem studies revealed gastrointestinal scleroderma as the morphological manifestation of her malabsorption syndrome, ocular and skeletal myopathy with ragged red fibers, peripheral neuropathy, vascular abnormalities of meningeal and peripheral nerve vessels. Biochemical examination of the liver and muscle tissues revealed a partial defect of cytochrome-c-oxidase (complex IV of the respiratory chain). This mitochondrial multisystem disorder may represent a separate entity to be classified between the spectrum of myoencephalopathies and oculo-gastrointestinal muscular dystrophy.

Expression of Endogenous Receptors for Neoglycoproteins, Especially Lectins, That Allow Fiber Typing on Formaldehyde-fixed, Paraffin-embedded Muscle Biopsy Specimens . A Glycohistochemical, Immunohistochemical, and Glycobiochemical Study'

A panel of biotinylated (neo)glycoproteins was used for specific detection of endogenous sugar receptors, especially lectins, in formaldehyde-fixed, paraffin-embedded muscle biopsy specimens from human deltoid, quadriceps, and biceps muscles, tibial and quadriceps muscles of rat, and bovine masseter muscle. The glycohistochemical probes used consisted of conjugates of a labeled, histochemically inert carrier protein and various covalently linked, histochemically crucial sugar moieties. Specific binding of alpha-L-fucoside, beta-D-galactoside, beta-D-xyloside, and alpha-D-mannoside to muscle sections was detected, showing no species-specific differences. The presence of receptors for the N-acetylated sugars in natural glycoconjugates, and for sugars with a phosphate group, i.e., mannose-6-phosphate and galactose-6-phosphate, was demonstrated glycohistochemically. However, these binding specificities revealed species-specific differences, e.g., the absence of N-acetyl-D-galactosamine-specific receptors or galactose-6-phosphate-specific receptors in rat muscle. Other charged sugars included glucuronic acid and sialic acid, which bound only to ox and rat muscle or failed to reveal their respective receptors in all types of muscle investigated. This different extent of staining with anionic probes served as a further control to ascertain carbohydrate binding specificity. Positive glycohistochemical reaction developed within sarcomeres only at the level of A-bands. Granular staining was observed in the sarcoplasm among the myofibrils and also in the subsarcolemmal regions. Differences in expression of glycohistochemically detectable sugar receptors were noted between type 1, type 2A, and type 2B fibers. The molecular properties of one type of glycohistochemically detectable sugar receptor were inferred both immunohistochemically and biochemically. An antiserum against an endogenous beta-galactoside-specific lectin from muscle tissue localized this lectin within sections consistently similar to (neo)glycoproteins, detecting beta-galactoside-specific receptor(s). This similarity of binding patterns strongly supports the assumption that (neo)glycoproteins with beta-galactoside termini indeed bind to the respective endogenous lectin. The lectin-specific antiserum enabled us to ascertain that glycohistochemical fiber typing corresponds to enzyme histochemical typing. Moreover, biochemical purification using affinity chromatography and subsequent affinity elution revealed only the immunohistochemically detectable beta-galactoside-specific lectin. Consequently, use of a panel of neoglycoproteins, when frozen sections for histochemical analysis are not available, co

Heparin Binding Lectin of Human Placenta as a Tool for Histochemical Ligand Localization and Isolation

Biotinylated heparin has been used to detect the presence of specific binding sites in sections of human placenta, which has prompted demonstration of expression of lectin activity for this proteoglycan. Purification of this lectin from full-term placenta facilitates the synthesis of its biotinylated derivative, using biotin-amidocaproyl hydrazide, without affecting its activity. It also enables immunization to obtain antibodies. The labeled lectin is shown to bind specifically to nuclear and cytoplasmic locations in various cell types of human placenta, nuclear expression of lectin binding sites being more pronounced at the full-term stage than after 8 weeks of development. The structurally related histone H2B exhibits obvious differences in its binding pattern. The presence of ligands accessible to the lectin whose binding activity can be inhibited by addition of an excess of heparin correlates in most instances with the level of lectin expression detected immunohistochemically. Biochemical information on the nature of the glycohistochemically inferred lectin-specific ligand(s) is obtained by affinity chromatography on resin-immobilized lectin. It leads to isolation of a proteoglycan with similar electrophoretic mobility in agarose-polyacrylamide gel electrophoresis relative to the independently purified heparan sulfate-containing fibronectin binding proteoglycan from human placenta. Both fractions inhibit binding of heparin to the lectin and contain immunologically detected co-purified lectin, emphasizing their ligand properties. Application of labeled tissue lectins in conjunction with lectin-specific antibodies is proposed to obtain valuable insights into the expression of the receptor as well as the ligand part of protein-carbohydrate recognition.

Comparative Histochemical and Biochemical Analysis of Endogenous Receptors for Glycoproteins in Human and Pig Peripheral Nerve

Abstract: Endogenous sugar‐binding proteins were localized in sections of human and pig peripheral nerves by the application of two types of labelled ligands: neoglycopro‐teins (chemically glycosylated carrier proteins that had proven to be histochemically inert) and desialylated, naturally occurring glycoproteins. These proteins allowed evaluation of the presence and distribution of endogenous receptors for carbohydrates, commonly present in cellular glycoconjugates. (Neo)glycoprotein binding was similar, but not identical, for the two types of mammalian peripheral nerves. The pig nerve differed from the human nerve in more pronounced staining when using different types of β‐galactoside‐terminated (neo)glycoproteins and charge‐carrying neoglycoproteins, such as bovine serum albumin, bearing galactose‐6‐phosphate residues, glucuronic acid residues, and sialic acid residues. Comparative biochemical analysis of certain classes of sugar receptors by affinity chromatography and gel electrophoresis revealed the presence of sugar receptors that can contribute to the histochemical staining in a pattern with certain significant differences among rather similar expression for the two species. The assessment of sugar receptor distribution by application of (neo)glycoprotein binding among morphologically defined regions in nerves may hold promise in detecting developmental regulation and changes during nerve degeneration and subsequent regeneration after trauma or pathological states. Correlation of these results to changes in the structure and abundance of glycoconjugates, which are the potential physiological ligands of endogenous sugar receptors commonly detected by plant lectins, may help to infer functional relationships.

Identification of a cell cycle-dependent gene product as a sialic acid-binding protein

A Ca2+-dependent sialic acid-binding protein was purified on fetuin-Sepharose from various types of human tissue. The molecular mass was determined to be 10,315 Da by laser desorption mass spectrometry. Partial sequence analysis after cyanogen bromide cleavage that yielded one N-terminus accessible for Edman degradation revealed an identity to an internal stretch following the only methionine residue within a putative amino acid sequence (Mr 10,048), deduced from the cDNA of a cell cycle-specific gene. The reported biochemical identification is a prerequisite to infer the biological role of the so far undetected gene product. Initial glycohistochemical studies with sialic acid-(BSA-biotin) raised evidence for nuclear localization of sialic acid-binding sites that might reflect, at least in part, detection of this protein.

Regional differences in the distribution of endogenous receptors for carbohydrate constituents of cellular glycoconjugates, especially lectins, in cortex, hippocampus, basal ganglia and thalamus of adult human brain

SummaryTen different types of labelled neoglycoproteins, exposing glycohistochemically pivotal carbohydrate moieties that mostly are constituents of naturally occurring glycoconjugates with an aromatic spacer, were synthesized. The panel was applied to fixed, paraffin-embedded sections of different cortical regions and white matter, of hippocampal gyrus, basal ganglia, thalamus nuclei and adjacent areas of adult human brain to comprehensively map the presence of respective binding sites in these parts. Compliance with accepted criteria for specificity of binding was routinely ascertained. Overall, not a uniform binding pattern, but a distinct distribution with regional differences on the level of specific cytoplasmic and nuclear staining in nerve cells was determined, fiber structures being generally labelled with medium or strong intensity. For example, among the neurons localized in the five cortical laminae the binding of N-acetyl-d-galactosamine varied from strong to undetectable. Biochemical analysis, employing carbohydrate residues as affinity ligands in chromatography, proved that the neuroanatomically different regions exhibited a pattern of receptors with notable similarities. These results on endogenous binding sites for glycoconjugates, especially lectins, are complementary to assessment of localization of cellular glycoconjugates by plant lectins and carbohydrate-specific monoclonal antibodies. They are thus a further obligatory step to substantiate the physiological roles of recognitive protein-carbohydrate interactions in the central nervous system.

Endogenous carbohydrate-binding proteins in oligodendrogliomas

SummaryDifferentiated and anaplastic oligodendrogliomas revealed intracytoplasmic and nuclear presence of endogenous carbohydrate-binding proteins by application of labelled (neo)glycoproteins in histochemical analysis. The histochemical patterns showed differences between the differentiated and anaplastic forms of the same tumor type. Xylose-, lactose- and asialofetuin-specific carbohydrate-binding proteins could be detected in both types of tumors with the same staining intensity. However, maltose-specific carbohydrate-binding proteins were present only in the differentiated form. An inverse intensity of the histochemical reaction was observed with galactose-6-phosphate-, galactose-β(1.3)-N-acetylglucosamine-N-acetylglucosamine-and mannose-(BSA-biotin) and fucose-(BSA-biotin) respectively, when the differentiated and anaplastic oligodendrogliomas were compared with each other. These differences document changes in the pattern of histochemically detectable carbohydrate-binding proteins, suggesting a role for endogenous carbohydrate-binding proteins in the tumor cell differentiation. These data indicate the potential usefulness of labelled (neo)glycoproteins as a new type of marker for histopathological diagnosis.

(Neo)glycoproteins as tools in neuropathology: histochemical patterns of the extent of expression of endogenous carbohydrate-binding receptors, like lectins, in meningiomas.

SummaryBiotinylated (neo)glycoproteins were used to specifically detect endogenous sugar receptors such as lectins in sections of formaldehydefixed, paraffin-embedded tissue from meningiomas. The histochemical methods used consisted of the application of a carrier protein and various covalently linked sugar moieties, available mainly through chemical synthesis, in an optimized standard protocol. They proved valuable in elucidating differential binding patterns within the various meningioma subtypes.α-Fucoside-, β-galactoside-, α-mannoside-and β-xyloside-specific carbohydrate-binding receptors were detected in all the tumor subclasses examined, although the levels of expression exhibited pronounced quantitative differences. In addition, differences in the extent of histochemical staining were observed, using a labelled carrier protein, derived from N-acetylglucosamine and mannose-6-phosphate moieties, respectively. Quantitative differences in the reaction intensity were also measured in the respective subtypes. Receptors for N-acetyl-D-galactosamine were detected only in the anaplastic forms, while glucuronic acid-specific receptors were only present in the meningotheliomatous meningioma. In contrast to the other types, malignant meningiomas failed to show cytoplasmic staining with the α-glucoside-specific maltose-(BSA-biotin). Distinct differences in the pattern of expression of endogenous sugar receptors, evaluated by a standard protocol, provided further evidence for a possible additional subtype of men-ingioma, the submalignant meningioma. Our results suggest that labelled (neo)glycoproteins could be used routinely as tools for assessing the expression of endogenous sugar receptors in diagnostic neuro-oncology.

The ultrastructure of normal and denervated human facial muscle.

The ultrastructure of normal human facial muscles from 25 nonparalytic and 17 paralytic patients revealed normal features in nondenervated human facial muscles, identical to the fine structure of other normal human and mammalian cross-striated muscle fibers. However, in denervated facial muscle, a broad spectrum of ultrastructural lesions had affected sarcomeres, abnormal inclusions, and organelles. A large variety of inclusion bodies, some of which have not been described, were also found. The spectrum of ultrastructural changes showed no dependence on the length of the denervation period. There were no inclusion bodies in all the normal facial muscle biopsies. To our knowledge, this study represents the first systematic electron microscopic investigation of normal and denervated human facial muscles.

Nebulin and titin expression in Duchenne muscular dystrophy appears normal

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5′‐region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.