Accumulation Of Storage Material Research Articles

2 Cellular pathology and pathogenic aspects of neuronal ceroid lipofuscinoses

Lysosomal accumulation of autofluorescent, ceroid lipopigment material in various tissues and organs is a common feature of the neuronal ceroid lipofuscinoses (NCLs). However, recent clinicopathologic and genetic studies have evidenced that NCLs encompass a group of highly heterogeneous disorders. In five of the eight NCL variants distinguished at present, genes associated with the disease process have been isolated and characterized (CLN1, CLN2, CLN3, CLN5, CLN8). Only products of two of these genes, CLN 1 and CLN2, have structural and functional properties of lysosomal enzymes. Nevertheless, according to the nature of the material accumulated in the lysosomes, NCLs in humans as well as natural animal models of these disorders can be divided into two major groups: those characterized by the prominent storage of saposins A and D, and those showing the predominance of subunit c of mitochondrial ATP synthase accumulation. Thus, taking into account the chemical character of the major component of the storage material, NCLs can be classified currently as proteinoses. Of importance, although lysosomal storage material accumulates in NCL subjects in various organs, only brain tissue shows severe dysfunction and cell death, another common feature of the NCL disease process. However, the relation between the genetic defects associated with the NCL forms, the accumulation of storage material, and tissue damage is still unknown. This chapter introduces the reader to the complex pathogenesis of NCLs and summarizes our current knowledge of the potential consequences of the genetic defects of NCL-associated proteins on the biology of the cell.

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos.

A mouse gene knockout model for juvenile ceroid-lipofuscinosis (batten disease)

The human hereditary ceroid-lipofuscinoses are a group of autosomal recessively inherited diseases characterized by massive accumulations of autofluorescent lysosomal storage bodies in the cells of many tissues and by neuronal degeneration throughout the central nervous system. There are a number of clinically and genetically distinct forms of ceroid-lipofuscinosis, the most common of which is the juvenile type, also known as Batten disease and CLN3. To study the mechanisms that lead to pathology in CLN3 and to evaluate potential therapies, a mouse model has been generated by targeted disruption of the mouse ortholog of the CLN3 gene (Cln3). As in affected humans, mice homozygous for the disrupted Cln3 allele show accumulation of autofluorescent storage material in neurons and other cell types. The storage material consists of membrane-bounded intracellular inclusions with ultrastructural features typical of the ceroid-lipofuscinoses. The accumulation of this storage material validates the Cln3 knockout mice as a model for the human disorder.

Normal superoxide radical production in the neuronal ceroid-lipofuscinoses.

The neuronal ceroid-lipofuscinoses (NCL) are a group of inherited progressive encephalopathies. Striking histomorphological feature of the NCL is the accumulation of storage material within the lysosomes in neural and extraneural cells. The basic underlying defect causing the disease is not known. Presupposing a disturbance in lipid peroxidation, some authors recommend antioxidant treatment to slow down the progression of the disease. In this study, the superoxide radical production of polymorphonuclear leukocytes as one potential source of reactive oxygen species was measured in this disorder for the first time. No significant difference in this production between affected individuals and healthy controls could be detected. Our findings cast doubt on the therapeutic benefit of antioxidant treatment.

Interrelationships between ultrastructure, sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation and deacclimation in poplar ( Populus × canadensis Moench ‹ robusta›) Wood

Summary Ray parenchyma cells of poplar wood ( Populus × canadensis Moench ‹ robusta ›) were studied for changes in ultrastructure and in the level of various sugars in fall, winter and spring in relation to freezing resistance as measured by LT 50 (temperature at which 50% of the cells are injured). Before acclimation (mid-October), frost hardiness is low (LT 50 −3 to −5 °C), sugar levels have not yet increased while the ray cell protoplast has already become dense in structure. This is due mainly to the elimination of large vacuoles, their replacement by small storage protein vacuoles, and the vast accumulation of storage material in general, i.e. in amyloplasts, protein bodies, and oleosomes. Frost acclimation started during leaf fall (end of October) concomitant with a sudden increase of total sugar content, consisting mainly of sucrose and its galactosides. An increase in these sugars is paralleled at the ultrastructural level by a prominent vesicular and cisternal ER-system originating in the cytoplasm. Full acclimation (LT 50 lower than −50 °C) is obtained within 3 weeks (mid-November) and coincides with maximum content of sucrose and its galactosides (reaching 300 µg mg −1 DW in ray cells) and with a prominent development of vesicular and cisternal ER. The evidence for the accumulation in this elaborated ER-system of these sugars is discussed and a proposal for its functional significance both as an intrinsic device for dehydrating the protoplast during frost hardening and for delivery of membrane material to the plasma membrane is made. Transient rises of winter temperature above freezing were paralleled by a noticeable decrease in frost resistance (LT 50 −40 to −30 °C), declining sugar levels, and by gradual diminution of the particular ER-vesicles in cytoplasm. In March, at temperatures of 5 to 10 °C, sugar content rapidly lowers concomitandy with complete vanishment of vesicles from the cells. A decrease in frost resistance, in contrast, begins later, e.g. in mid-April, suggesting the involvement in frost hardiness of non-sugar compounds at this stage.

Rhodobacter sphaeroides RV cultivation and hydrogen production in a one- and two-stage chemostat

Rhodobacter sphaeroides RV cultivation and hydrogen production were studied in a one- and two-stage chemostat using lactic acid as substrate. Light saturation was observed when light intensities equal to or above 10 klx were applied. Under light saturation, the two-stage chemostat appeared to be very effective for hydrogen production, allowing complete nitrogen removal by bacterial growth in the first reactor. The hydrogen evolution rate in the second reactor was up to 75 ml H2 (g dry weight)−1 h−1. Accumulation of storage material was observed in the second reactor of the two-stage chemostat under a large carbon excess and limiting light irradiance. The optimal hydraulic residence time was 15 h for both stages, leading to a total hydrogen production about 40% higher than in the one-stage chemostat. Under increasing influent ammonium and yeast extract concentrations, opposite trends of decreasing bacterial activity and increasing concentration resulted in a linear increase of the overall hydrogen production to 1.4–1.6lH2 (l reactor)−1 day−1. Hydrogen production quickly fell when nitrogen was not completely metabolised. The hydrogen evolution rate was also found to depend on lactic acid concentration, and maximum bacterial activity was observed at 100 mM influent lactic acid.

Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

ABSTRACTWater deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post‐mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post‐fertilization period. Greenhouse‐grown maize was subjected to water‐limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical‐region kernels, but had little effect on middle‐region endosperms. Rewatcring did not allow cell division to recover in apical‐region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical‐region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A‐content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA‐content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.

Mitochondrial damage results in a reversible increase in lysosomal storage material in lymphoblasts from patients with juvenile neuronal ceroid-lipofuscinosis (Batten Disease).

We have previously demonstrated reduced phospholipid fatty acid content in blood cells and cultured skin fibroblasts from patients with JNCL. This has led to an experimental treatment regimen consisting of dietary supplementation with polyunsaturated fatty acids (PUFAs). In order to study the effects of PUFA supplementation in vitro, we have developed a laboratory model based upon cultured lymphoblast cell lines. We have transformed lymphocytes from four JNCL patients in whom disease linkage to chromosome 16 was informative. Cells from patients and controls were cultured with and without antibiotic (50 micrograms/ml gentamycin) and with and without PUFA supplementation. None of the control cells demonstrated significant storage under any of the above conditions. In gentamycin treated cells, we observed that many of the mitochondria were damaged. In addition, cells from patients incubated with gentamycin demonstrated large accumulations of autofluorescent storage material. Disease cells grown in the presence of antibiotic and PUFAs did not demonstrate a significant accumulation of storage material; this suggests a direct relationship between mitochondrial damage and storage of autofluorescent material. Moreover, it appears that this storage (but not mitochondrial damage) is reversed by the addition of PUFAs.

The Accumulation of Storage Materials in Ray Cells of Poplar Wood ( Populus x canadensis ): Effect of Ringing and Defoliation

Summary The accumulation of individual storage compounds, i.e. starch, sugars, protein and fat, was followed under normal conditions and under the influence of ringing and defoliation in the ray parenchyma of poplar twigs (Populus x canadenis Moench ). Under normal conditions, the individual compounds become deposited at clearly different periods of the year: starch accumulates continuously from late spring until fall, protein primarily during the short period of yellowing of leaves in fall, and fat mainly in late summer. Highly increased sugar levels are found from late fall until the end of winter. By ringing twigs ( = photosynthate surplus conditions), protein deposition can be induced already in summer and the accumulation levels of starch, fat and protein also can be increased considerably above those of controls. Defoliation in early summer, on the other hand, leads to an initial decrease, but then with the aid of a new set of leaves to an almost unaltered storage material deposition until fall. In contrast to starch, fat and protein levels, however, become lower. It is concluded (i) that deposition of storage protein in the ray parenchyma is not directly dependent on the photoperiod per se, (ii) that the accumulation levels of various storage compounds normally found in the tissue are not at their maximum levels but can be increased significantly, and (iii) that their deposition is intimately related to the actual levels of photosynthates available.

Co-factor regeneration in the production of 1,2-epoxypropane by Mycobacterium strain E3: the role of storage material

When grown on ethene Mycobacterium strain E3 produces epoxyalkanes from alkenes in an oxygen- and NADH-dependent reaction. The process of co-factor regeneration was studied by analysing the intracellular pools of NADH and storage material during the production of 1,2-epoxypropane from propene. With the depletion of NADH the production of 1,2-epoxypropane stopped. NADH could be regenerated from the oxidation of added co-substrate or from oxidation of storage material. Cells cultivated in chemostat culture under nitrogen limitation produced more 1,2-epoxypropane compared to cells cultivated under carbon limitation, due to their higher content of storage material. Addition of glucose to cells grown under carbon limitation stimulated the formation of 1,2-epoxypropane. The uptake of glucose resulted in the accumulation of storage material, which was utilized after depletion of the glucose. Glycogen and trehalose were the preferred forms of storage material used for co-factor regeneration. From the results it was concluded that formation and utilization of storage material play a crucial role in the process of co-factor regeneration in Mycobacterium strain E3.

Altered protein patterns in brains of children with neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative diseases characterized by massive intralysosomal accumulation of storage materials. We have studied the protein patterns in 5 NCL, 5 control, and one Alzheimer disease brains. When protein patterns in NCL and control brain gray matter homogenates were examined by SDS-PAGE, NCL brains showed an absence or greatly reduced amounts of the Mr 160-180 kDa component and reduced amounts of the Mr 29-36 kDa component. Concomitantly, an increase in several components with Mrs of 45-50 kDa was noted. The 180 kDa polypeptide appears to be a glycoprotein because it was bound to the lectins concanavalin A and Ulex europaeus. Recently, the abnormal processing of amyloid protein precursor (APP) and its potential role in NCL have been suggested. Possible defects in tissue proteases and protease inhibitors may be considered responsible for the presence of these amyloid beta protein precursor fragments. To examine this possibility we are using polyclonal antibodies to the C terminal 672-695 (APP) and monoclonal antibodies to inter-alpha-trypsin inhibitor. Polypeptides with molecular weights of approximately 35-38 kDa were detected in the NCL brain, but not in controls in both cases. These findings suggest abnormal protein processing in NCL brain tissue, disturbances in protein and glycoconjugate metabolism, impaired lysosomal function (i.e., metabolic enzyme and/or proteases/proteinase inhibitor abnormalities), and the involvement of improperly processed APP.

Accumulation of Storage Materials, Precocious Germination and Development of Desiccation Tolerance During Seed Maturation in Mustard (Sinapis alba L.)

AbstractUnder defined environmental conditions (20°C, continuous light of 15 klx) development of mustard seeds from artificial pollination to maturity takes about 60 d. After surpassing the period of embryo cell division and histodifferentiation (12–14d after pollination = dap), the seed enters into a maturation period. The time courses of various physiological, biochemical, and structural changes of embryo and testa during seed maturation were analyzed in detail (dry and fresh mass changes, osmotic and water potential changes, respiration, DNA amplification by endomitosis, total ribosome and polysome formation, storage protein synthesis and accumulation, storage lipid accumulation). In addition to the final storage products protein and lipid, embryo and testa accumulate transiently large amounts of starch within the chloroplasts during early maturation. Concomitantly with the subsequent total breakdown of the starch, the plastids lose most of their internal structure and chlorophyll and shrink into proplastids, typical for the mature seed. At about 30 dap the seeds shift from a desiccation‐sensitive to a desiccation‐tolerant state and are able then to germinate rapidly upon drying and reimbibition. If isolated from the immature fruit and sown directly on water, the seeds demonstrate precocious germination from about 13 dap onwards. Young seeds (isolated ≦ 38 dap) germinate only after surpassing a lag‐phase of several days (after‐ripening) during which the embryo continues to accumulate storage protein and lipid at the expense of the surrounding seed tissues. We conclude from these results that the maturing seed represents a rather closed developmental system which is able to continue its development up to successful germination without any specific regulatory influence from the mother plant. Immature seeds are able to germinate without a preceding dehydration treatment, which means that partial or full desiccation does not serve as an environmental signal for reprogramming seed development from maturation to germination. Instead, it is argued that the water relations of the seed are a critical element in the control of maturation and germination: during maturation on the mother plant the embryo is subject to a considerable turgor pressure (of the order of 12 bar) accompanied by a low water potential (of the order of −12 bar). This turgor permits maturation growth but is subcritical for germination growth. However, upon imbibition in water, the low water potential provides a driving force for a burst of water uptake overcoming the critical turgor threshold and thereby inducing germination.

Narrow trachea in mucopolysaccharidoses.

Nine of 56 patients with mucopolysaccharidoses (MPS) showed small tracheal diameters on their frontal chest radiographs. Autopsy of an MPS I-H (Hurler disease) patient demonstrated that the small calibre was secondary to deposition of glycosaminoglycan (mucopolysaccharide). Autopsies of two patients with other storage diseases, one with geleophysic dysplasia and one with mucolipidosis II, also exhibited compromise of their airways because of storage material accumulation.

Diagnosis of late infantile neuronal “ceroid-lipofuscinosis” from histochemical and ultrastructural changes in dental pulp

Sections of dental pulp were removed from a tooth of a boy 3 years 10 months of age with myoclonic seizures. When stained with hematoxylin and eosin, these sections showed eosinophilic inclusions. Ultrastructurally, these inclusions were large cytosomes filled with whorls of electron-opaque substance. The opaque material consisted of bilaminate strands, approximately 15 nm in diameter, arranged in a curvilinear pattern compatible with that seen in the late infantile form of “ceroid-lipofuscinosis.” Most histochemical staining reactions were compatible with ceroid or lipofuscin, with the exception of the diamine silver reaction. Late infantile ceroid-lipofuscinois represents another disease in which asymptomatic accumulation of storage material occurs in the dental pulp.

Ultrastructural study on nervous system of fetus with GM1-gangliosidosis type 1.

The nervous system of a 22-year-old fetus with GM1-gangliosidosis type 1 was studied by electron microscopy. The tissues thus examined were the cerebral cortex at the parietal region, the cerebellum, the thoracic spinal cord, the Auerbach's myenteric plexus in the large intestine and the radial nerve fibers. In the cerebral cortex, membrane-bound vacuoles, which occasionally contained stacks of fine fibrils, were observed in the large young neurons in the deeper part of the cortical plate. The neurons in the other part of the cerebral cortex carried no storage materials. In the cerebellum, the membrane-bound vacuoles with stacks of fine fibrils were seen only in the Purkinje cells. The neurons in the spinal cord also contained several zebra-like bodies and the above membrane-bound vacuoles. As for the peripheral nervous system (PNS), neurons in the Auerbach's myenteric plexus carried membranous cytoplasmic bodies and zebra-like bodies. Some of the axons in the radial nerve fibers also contained a lot of pleomorphic electron-dense bodies and a few membranous cytoplasmic ones. These results show that the accumulation of storage materials is started in the large neurons which are produced in the early stage of neurogenesis in the central nervous system (CNS). Additionally, the observed membrane-bound vacuoles are considered to be structures which occur before the membranous cytoplasmic bodies and/or the zebra-like bodies. It is also elucidated that the PNS is affected earlier than the cerebral and cerebellar cortices and thoracic spinal cord.

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts.

Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity if the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing.

Life-Cycle Variation in Geophytes

Life-Cycle Variation in Geophytes

Estrogen-induced ultrastructural changes in cultured chick embryo hepatocytes

Administration of estradiol-17-beta induces the synthesis of vitellogenin in primary cultures of chick embryo liver. The ultrastructural changes accompanying steroid induced vitellogenin synthesis were investigated in hepatocytes cultures incubated for 0, 12 and 24 h following hormonal treatment. Both electron microscopy and immuno-chemical techniques were used. The immuno-fluorescence data indicate that the cultures contain a cell population of 90--95% hepatocytes, both in control and estradiol-treated groups. Ultrastructurally, cultured hepatocytes are similar to in vivo ones, except for a reduced accumulation of storage materials, glycogen and lipids. In the estradiol-treated cultures hepatocytes show an accumulation of free ribosomes, enlargement of rough endoplasmic reticulum, and development of the Golgi apparatus.

Utilization of Photosynthates for Growth, Respiration, and Storage in Tops and Roots of Lolium multiflorum

Abstract Lolium multiflorum L. was grown in pots in controlled environments. CO2‐exchange rates were continuously measured on two pots during 46 and 52 days, respectively, separating between tops and roots. After 20 days, the plants were entirely defoliated and the plants were then followed during the regrowth period. During the experiment, alternating 2–3 day periods of high and low irradiance were applied. Analogously treated plants were frequently harvested to obtain the distribution of assimilates between tops and roots. From integration of CO2‐exchange rates, diurnal photosynthesis and respiration were obtained, and utilization of assimilates was analysed.The respiration associated with the synthesis of new structural material (growth respiration) was dependent on assimilates originating from both the current and the preceding 24 h diurnal cycles. The amount of new structural material synthesized during the current 24 h diurnal cycle was estimated from the relative contribution of assimilates accumulated from the preceding and the current 24 h and diurnal cycles to growth respiration of the current 24 h. From this approximation, the respiratory components connected to synthesis of new structural material and to maintenance of already established material were found.Growth and maintenance respirations of the tops were alike during the predefoliation and the regrowth periods. For the roots, however, growth respiration was higher and maintenance respiration lower in the regrowth period. The difference between daily integrated CO2‐exchange and the amount converted into new structural material was assumed to be the daily change in assimilates stored. On the first day of a period of high irradiance, the assimilation per unit leaf weight was higher than on the following day of high irradiance, and an accumulation of storage material took place. On the first day of a period of low irradiance, the assimilation per unit leaf weight was lower than on the following day of low irradiance, and there was a depletion of assimilates stored. These effects were most pronounced during the regrowth period, indicating a change in the metabolic sink demand. This indicates a strong feedback mechanism between sources and sinks, in the sense that accumulation of products will inhibit assimilation.

Communicating hydrocephalus in children with genetic inborn errors of metabolism.

The authors have seen eight cases of communicating hydrocephalus in children with genetic metabolic disorders, namely, one mucopolysaccharidosis I (MPS I or Hurler syndrome), one MPS II (Hunter's disease), four MPS III (Sanfilippo syndrome) two of which were siblings, and two achondroplasias. The authors recommend surgical treatment of the latter but are doubtful about the former in which case hydrocephalus is only a contributing cause to severe dementia. In MPS hydrocephalus is due to accumulation of storage material in the piaarachnoid, as indicated by RISA cisternography carried out in six cases.