Lectin histochemical study of lipopigments with special regard to neuronal ceroid-lipofuscinosis. Results with concanavalin A

Concanavalin A (ConA) binding to lipopigments (LPs) of the lipofuscin type was proved to be due to the high content of mannose. The nature of the mannose bearing compound was twofold. One part was soluble in modified chloroform-methanol-water mixture (10:10:3) corresponding possibly to the oligosaccharyl diphosphodolichol (oligo-PP-Dol) described to be increased in LPs especially of inherited types. The second part, most probably a glycoprotein (GP), was entirely resistant to various extraction procedures. The ratio of the two components varied. The deposition of the typical lipofuscin (age pigment) was dominated by the GP component. Its amount was greatest in neurolipofuscin (especially in the olivary nucleus) and in the myocardium but very little in hepatocytic lipofuscin. In human neuronal ceroid lipofuscinoses (of early juvenile, and juvenile types) both components were found in large quantities in the storage granules of the affected neurons. The "protein type variant" of the storage material (Elleder 1978) displayed the highest degree of lipid-bound mannose accumulation, the GP component being extremely low or entirely absent. In the late infantile, infantile and Kufs variants studied in paraffin sections only, the GP component was detectable, too as in the case of the secondary neuronal LP in mucopolysaccharidoses and gangliosidoses. In the dog model of NCL lipid bound mannose clearly predominated, the GP component being concentrated in the cytoplasm and on the periphery od some storage granules. The nature of the GP component, a new finding of LP analysis, is discussed. The metabolic relationship between the two components is uncertain. Neither could be identified as the component resposible for autofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin histochemical study of olfactory neurons in the eel

Lectin histochemical studies were performed on paraffin embedded sections of the olfactory system of the eel to identify specific glycoconjugates on the surface of primary olfactory neurons. The olfactory receptors, the olfactory nerve fibres and their terminals in the bulbs were labelled with the lectins (SBA, BSA-I, BSA-I-B4 and DBA) HRP-conjugated or biotinylated. The lectin staining patterns indicate that the membrane of olfactory neurons of the eel had oligosaccharides with alpha-galactose and alpha-N-acetyl-D-galactosamine residues. These findings represent the demonstration of a molecular probe that recognizes specific sets of neurons. The identical histochemical features previously described in the olfactory neurons in amphibians suggest that these carbohydrate moieties might to related to modulation of the cell-cell interactions in the olfactory system of vertebrates.     

Electron microscopic study of immunoreactive LHRH perikarya with special reference to neuronal regulation

Summary In early postnatal rats, immunoreactive LHRH perikarya in the preoptic area were studied by light and electron microscopy. Synaptic junctions were found between the immunoreactive perikaryon or its process, and the immunonegative nerve fibers. The significance of these synapses is discussed in relation to possible mechanisms by which the activities of LHRH neurons are regulated.     

An autoradiographic study of the chromosomes of the rat,with special regard to the sex chromosomes

The pattern of terminal DNA synthesis of rat chromosomes was studied by means of tritiated thymidine incorporation and autoradiography. In the female, an entire X-chromosome underwent relative out-of-phase replication. In the male, the Y-chromosome showed a remarkable late replication. Several pairs of autosomes exhibited characteristic replicating patterns which were useful for their identification. The relation between the less prominent out-of-phase replication of the late replicating X-chromosome and the difficulty in finding the sex dimorphism of interphase nuclei of the rat is discussed.Contribution No 710 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo.One of the authors, S. Makino, is much delighted to dedicate this paper to Professor Dr. J. Seiler in celebration of his 80th anniversary, on the 16th May, 1966.     

Site dependent time optimization of protein synthesis with special regard to accuracy

The efficiency of protein synthesis is determined by its rate, accuracy, and energy consumption. With the energy consumption fixed, we optimize the system with respect to time and accuracy. Using an analytic model for a simple system and computer simulations for more complex systems, where also the possibility of errors is included, we demonstrate how different parts of the messenger RNA influence the protein production rate differently. The first part of the coding sequence is of major importance, since the availability of empty initiation sites is crucial, and queuing back to that region may interfere with initiation. The elongation rate at different positions depends on codon usage, on the concentrations of substrate and co-factors, and on the kinetic rate constants, including those of the proofreading branch(es). Ribosomal proofreading is a time consuming process and by allowing for more errors in the beginning of a protein, it is possible to increase the production rate of that protein. We calculate the mean translation time per functioning protein for various translation accuracies, and discuss the different strategies open to living cells.     

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.     

Fractionation of nucleic acids with special regard to rapidly labeled RNA.

Fractionation of nucleic acids with potassium acetate is useful for studying the synthesis of ribosomal RNA and messenger RNA. By quantitative removal of 4 and 5 s RNA and of DNA, the detection of messenger RNA is facilitated.     

High levels of brain dolichols in neuronal ceroid-lipofuscinosis and senescence

Dolichols as unesterified alcohols were identified as significant components of lipid extracts from storage cytosomes isolated post-mortem from the brains of patients with the infantile, late infantile, and juvenile types of neuronal ceroid-lipofuscinosis (NCL). Very small amounts of dolichols were present in the corresponding subcellular fractions of non-NCL brains. The nuclear fraction from NCL cerebral cortex contained the highest dolichol content expressed per milligram protein or lipid, whereas the crude mitochondrial fraction was the richest in normal brain. Highly significant elevations of dolichol levels were found in human cerebral cortex of patients with NCL and Alzheimer's disease compared with age-matched controls, but the levels were normal in Pick's disease. In human non-NCL cerebral cortex, dolichols increased from 16 micrograms/g at age 5 to over 200 at age 81. Rat cerebral cortex showed a similar progressive increase in dolichol content with age. The high dolichol values in NCL, Alzheimer's disease, and senescence appears to be related to the increase of lipofuscin in brain. This is the first time a uniform biochemical abnormality has been found in all childhood forms of NCL, but the enzyme defect is still unidentified. It may lie on pathways where dolichols and retinyl compounds are recycled in Golgi membranes and derived organelles during the biosynthesis of glycoproteins.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Pancreatic islets subjected to different concentrations of glucose in vitro. A study with special regard to mitochondrial changes.

Isolated pancreatic islets of mice and gerbils were cultured for 6 days at low (2mM) or high (20mM) concentrations of glucose after which they were studied using qualitative and quantitative electron microscopy, histo- and microchemistry, and X-ray microanalysis. Compared with the islets cultured at high glucose, those subjected to low glucose exhibited enhanced succinate dehydrogenase activity, a decreased content of adenosine triphosphate, and an increased volume of B-cell mitochondria which often were rounded or oval.     

Exome-sequencing confirms DNAJC5 mutations as cause of adult neuronal ceroid-lipofuscinosis

We performed whole-exome sequencing in two autopsy-confirmed cases and an elderly unaffected control from a multigenerational family with autosomal dominant neuronal ceroid lipofuscinosis (ANCL). A novel single-nucleotide variation (c.344T>G) in the DNAJC5 gene was identified. Mutational screening in an independent family with autosomal dominant ANCL found an in-frame single codon deletion (c.346_348 delCTC) resulting in a deletion of p.Leu116del. These variants fulfill all genetic criteria for disease-causing mutations: they are found in unrelated families with the same disease, exhibit complete segregation between the mutation and the disease, and are absent in healthy controls. In addition, the associated amino acid substitutions are located in evolutionarily highly conserved residues and are predicted to functionally affect the encoded protein (CSPα). The mutations are located in a cysteine-string domain, which is required for membrane targeting/binding, palmitoylation, and oligomerization of CSPα. We performed a comprehensive in silico analysis of the functional and structural impact of both mutations on CSPα. We found that these mutations dramatically decrease the affinity of CSPα for the membrane. We did not identify any significant effect on palmitoylation status of CSPα. However, a reduction of CSPα membrane affinity may change its palmitoylation and affect proper intracellular sorting. We confirm that CSPα has a strong intrinsic aggregation propensity; however, it is not modified by the mutations. A complementary disease-network analysis suggests a potential interaction with other NCLs genes/pathways. This is the first replication study of the identification of DNAJC5 as the disease-causing gene for autosomal dominant ANCL. The identification of the novel gene in ANCL will allow us to gain a better understanding of the pathological mechanism of ANCLs and constitutes a great advance toward the development of new molecular diagnostic tests and may lead to the development of potential therapies.     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

A mutation in the CLN8 gene in English Setter dogs with neuronal ceroid-lipofuscinosis

A heritable neurodegenerative disease of English Setters has long been studied as a model of human neuronal ceroid-lipofuscinosis (NCL). Megablast searches of the first build of the canine genome for potential causative genes located the CLN8 gene near the q telomere of canine chromosome 37, close to a marker previously linked to English Setter NCL. Sequence analysis of the coding region from affected dogs revealed a T-to-C transition in the CLN8 gene that predicts a p.L164P missense mutation. Leucine 164 is conserved in four other mammalian species. The C allele co-segregated with the disease phenotype in a two-generation English Setter family in a pattern consistent with autosomal recessive inheritance. All four NCL-affected family members were C/C homozygotes and all four obligate carriers were C/T heterozygotes; whereas, 103 unrelated dogs were all T/T homozygotes. These findings indicate that the CLN8 T-to-C transition is the likely cause of English Setter NCL.     

Lectin histochemical identification of carbohydrate moieties in opossum chemosensory systems during development,with special emphasis on VVA-identified subdivisions in the accessory olfactory bulb

The changes occurring in the testicular lamina propria (LP) of the seasonal breeder Octodon degus were analyzed. Four groups of animals were studied using electron microscopic procedures. The animals in group I were reproductively active, whereas those in groups II, III, and IV were in periods of recrudescence, regression, and resting, respectively. These changes, observed in animals maintained under laboratory lighting conditions, resembled those known to be elicited in feral populations by natural photoperiods. Testicular changes in each group were monitored by calculating the gonadosomatic and spermatogenetic indexes, and by obtaining averages of the diameter of the seminiferous tubules and the height of the seminiferous epithelium. The LP of group I animals consisted of a basal membrane formed by two to four lamellae, inner and outer acellular layers containing moderate numbers of collagenous fibrils, and single or double layers of smoothly contoured myoid cells encircled by a discontinuous lymphatic epithelium. The LP of group IV animals differed considerably from the LP of group I animals: The inner acellular layer was enlarged, and the basal membrane appeared composed of a variable number of lamellae with numerous folds and indentations. Myoid cells were very irregular in contour and enveloped by a well-developed surface coating. The LPs of animals in groups II and III possessed relatively similar characteristics, which appeared to be intermediate between those of groups I and IV. The significance of these morphological changes in the LP are discussed in relation to testicular changes induced by photoperiod and other normal or pathological processes. © 1995 Wiley-Liss, Inc.