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Notizen: Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ∼30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [35S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [35S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [35S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.

Notizen: Abstract: Activities of six lysosomal enzymes in the cerebellum of jaundiced homozygous (jj) Gunn rats were examined from 5 to 20 days of life and compared with those in heterozygotes (j+). Significantly higher enzyme activities were first detected at 8 days. The jj/j+ activity ratios of all enzymes peaked at 15 days. The ratios of β-glycerophospha-tase, β-mannosidase, and acid lipase were only 1.3–1.7, whereas those of arylsulfatase and cathepsin were 2.0 and 3.1, respectively. The most striking increase in activity was observed with β-glucuronidase, the ratio of which was 8.4. These results indicate a selective increase in activities of certain lysosomal enzymes in the hypoplastic cerebellum of jj rats.

Notizen: Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC 12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC 12 cells after 3 days of culture was in the range of 90–190 kDa for the intact form (Kav= 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90–190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120–150 kDa and 120–190 kDa (Kav= 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav= 0.22 on Sephacryl S-300 [40–84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)]. The CS and core protein of this CSPG had apparent molecular masses of 26 kDa and 15–17 kDa, respectively. The peak I fraction was separated further into three fractions of different molecular sizes on Sephadex G-200 (I-1,I-2,I-3). In the presence of NGF, peak I-1 prepared after 3 days of culture consisted of mostly CSPG (130–170 kDa by SDS-PAGE; Kav= 0.13 on Sephacryl S-300) with CS of 32 kDa and a core protein of 105 kDa. Peaks 1–2 and 1–3 contained, respectively, CSPGs of 38–74 kDa and 31–56 kDa for their intact forms, with CSs of 25 kDa and 15 kDa. The apparent molecular masses of the core proteins of their CSPGs were 15–17 kDa. These CSPGs had similar molecular sizes, irrespective of the presence of NGF or the duration of culture. (4) These results indicate that structural changes in PGs during the neuritogenesis in PC12 cells induced by NGF occurred mostly in cell-associated PGs and preceded elongation of neurites.

Notizen: Developmental changes of cyclic nucleotides were studied in the hypoplastic cerebellum of jaundiced Gunn rats over the period of postnatal days 8 to 30. The mitogenic activity of glia maturation factor was also measured at day 15. In jaundiced homozygotes(jj), the amount of cyclic GMP on a protein basis was not significantly different from that in control heterozygotes (j+) at either day 8 or 15, but at day 30 it was reduced to about 19% of the control. On the other hand, a lowered nucleotide level on a wet weight basis in jj rats was already statistically significant at day 15. In contrast to cyclic GMP, the rates of increase of cyclic AMP on a wet weight basis were almost the same in the two groups of rats, but the nucleotide levels on a protein basis at days 15 and 30 were a little, but significantly, higher in jj rats than in j+ rats. The activity of glia maturation factor in jj rats was found to be 1.5-3 times as high as that in j+ rats. Possible implications of the present results are discussed.

Notizen: Abstract: Levels of the β-subunit of nerve growth factor (β-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. β-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified β-NGF reacted only with β-NGF. The assay for β-NGF was performed by incubation of F(ab′)2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab’ fragments labeled with β-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of β-NGF in almost all organs of mice. The amount of β-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3–5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of β-NGF (〉 100 pg/ml). The level of β-NGF at 4 months of age was relatively high (2–5 ng/g) in the pancreas, spleen, and ovary among the organs examined, excluding the submandibular glands. An unexpected finding was that levels of β-NGF not only in submandibular glands, but also in the brain, adrenal glands, and spinal cord, of males were three to seven times as high as levels in the analogous organs of females. Possible mechanisms regulating the level of β-NGF in neural and paraneural tissues are discussed.

Notizen: Abstract: PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PCI 2D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.

Notizen: The behavior of marker proteins of glial cells [α-enolase, β-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of eerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, γ-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of β-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in α- and γ-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of β-S100 and α-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of γ-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that β-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of eerebellar damage, respectively, but that a-enolase is less available.

Notizen: Abstract: The effect of bilirubin on the temperature dependence of Na, K-ATPase activity was investigated with NaI-treated microsomes prepared from young (13- to 23-day-old) and adult (about 1-year-old) rat cerebra. The Arrhenius plots for the enzymic activities in the young and adult rats showed break temperatures at 26 and 18°C, respectively. In the young rat enzyme, bilirubin caused a shift of the break temperature to 23 and 22°C at concentrations of 60 and 120 μM, respectively, with a significant increase of the activation energy below the break temperature. However, no significant changes in the break temperature and the activation energy were observed in the adult rat enzyme exposed to the same concentrations of the pigment. The results suggest that the lipid environment surrounding the enzyme and modulating its activity in the plasma membranes may differ between the young and adult rat cerebra, and that bilirubin may change the physical state of the lipids related to the activity of the young rat Na, K-ATPase.

Notizen: Abstract: We have investigated the postnatal development of cerebellar glutamate decarboxylase (GAD), acetylcholine esterase (AChE), 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), arylsulfatase, succinate dehydrogenase (SDH), Na,K-ATPase, arylesterase, and lactate dehydrogenase (LDH) activities in homozygous (jj) Gunn rats, comparing them with those in heterozygotes. Also examined were GAD activities in the anterior and posterior parts of the vermis cerebelli on day 13. The specific activities of GAD on the basis of wet weight (g) were lower in jj rats after day 10, and remained unchanged from days 10 to 15 in jj rats with severe hypoplasia. On day 13, GAD on wet weight and protein (mg) bases in the anterior part OfJJ rats was lower than that in the posterior parts of both rats. On the contrary, AChE activities based on wet weight after day 10 and on protein after day 5 in jj rats were significantly elevated. While correlations of GAD on a wet weight basis with the cerebellar wet weight were positive after day 8, those of AChE on wet weight and protein bases were inverse after days 10 and 5, respectively. CNPase and SDH activities based on protein in jj rats were higher after day 15 and showed inverse correlations with the cerebellar wet weight after days 15 and 10, respectively. Arylsulfatase activities on wet weight and protein bases in jj rats, which had a peak on day 20, were significantly high after days 10 and 8, respectively. Arylsulfatase activities by wet weight on days 10 to 20 and by protein after day 8 were inversely correlated with cerebellar wet weight in jj rats. These results suggest that the cerebellar inhibitory neurons, the axons of which make synaptic connections to the target cells from days 10 to 15, are selectively affected by bilirubin inji rats with severe hypoplasia. The enhanced arylsulfatase activity in jj rats on days 10 to 20 may be due to the increased number of lysosomes, suggesting that cell damage by bilirubin followed by cell destruction occurs. A high level of AChE activity in jj rats appears to show a relative increase in density of the mossy fibers in the hypoplastic cerebellum.

Notizen: On the basis of our previous findings that a 50,000-dalton protein (GR-50) shows a marked increase in the hypoplastic cerebellum of jaundiced homozygous Gunn rats and its electrophoretic behavior is similar to that of glial fibrillary acidic protein (GFAP), we tried to identify GR-50 as GFAP by two-dimensional electropho-resis of rat cerebellar membrane proteins using an improved immunoblotting method. In this method a blot immunostained for a specific antigen was also visualized for other proteins, thereby enabling us to determine the location of the antigen on the blot more precisely. With the methodology it was found that GFAP antigen occupied exactly the same position as GR-50 on the blot, suggesting strongly the identity of both proteins. Immunohistochemical studies revealed that in the cerebellum of homozygotes compared with that of heterozygotes GFAP antigen was greatly increased and that it was especially rich in the homozygous rat cerebellar lobules with a high degree of hypoplasia. Further, it was shown that not only the fibers of the Bergmann glial cells but also their somata were intensely immunostained in the affected lobules. A 47,000-dalton protein (SG-47), which has been reported to be increased in staggerer mutant mice with cerebellar hypoplasia, also immunoreacted with the antiserum to GFAP.