A possible contribution by glial cells to neuronal energy production enzyme-histochemical studies in the developing rat cerebellum

Summary Recent reports have revealed that certain neurons do not survive in vitro in the presence of glucose, which is the primary substrate and exclusive source of energy in the brain. But these neurons can survive in the presence of low-molecular-weight agents such as pyruvate, which are supplied by glial cells (Selak et al. 1984). To test whether this result also holds true in vivo, we investigated the distribution of hexokinase, lipoic dehydrogenase, -hydroxybutyrate dehydrogenase, and glucose-6-phosphate dehydrogenase activities in the developing rat cerebellum. Hexokinase activity was relatively higher in glial cells than in neurons. After postnatal day 8, the activity of hexokinase could hardly be detected in Purkinje cells, whereas it was highest in Bergmann glial cells. Purkinje cells were the only type of neuron with high levels of lipoic dehydrogenase at all ages tested. -Hydroxybutylate dehydrogenase activity was also high in Purkinje cells, especially in those from young rats. Relatively high glucose-6-phosphate dehydrogenase activity was demonstrated in basket and stellate cells from adult brain. Thus, it appears that, in vivo, certain neurons utilize relatively little glucose, and it is indeed possible that glial cells may supply some substance(s) other than glucose, for example pyruvate, as the primary source of energy.     

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Tissue Distribution and Immunocytochemical Localization of Neurotrophin-3 in the Brain and Peripheral Tissues of Rats

Abstract: The tissue distribution of neurotrophin-3 (NT-3) was investigated in rats at 1 month of age using a newly established, sensitive two-site enzyme immunoassay system for NT-3, as well as the immunocytochemical localization of this protein. The immunoassay for NT-3 enabled us to quantify NT-3 at levels > 3 pg per assay. In the rat brain, NT-3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT-3 was widely distributed in peripheral tissues. Appreciable levels of NT-3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT-3 bound to pyramidal cells in the CA2-CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT-3-immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT-3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT-3 not only functions as a classical target-derived neurotrophic factor but also can play other roles.     

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β-NGF levels disappeared. Average levels of β-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions. The concentrations of mRNA for β-NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain-derived neurotrophic factor and neurotrophin-3 between the two sexes. Thus, in some regions of the brain, the production of β-NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β-NGF in these region.     

1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions

Three species of microalgae able to produce eicosapentaenoic acid(EPA) were collected from brackish and sea water around Japan. The species were identified as Navicula saprophila, Rhodomonassalina and Nitzschia sp. EPA as a proportion of total fatty acids increased in the presence of acetic acid for Rhodomonas salina and Nitzschia sp. However, Navicula saprophila displayed the greatest productivity of EPA and the EPA content of its biomass was enhanced under mixotrophic conditions in the presence of acetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution of Brain-Derived Neurotrophic Factor in Rats and Its Changes with Development in the Brain

Abstract: A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.     

Dynamic sweating response of man to infrared irradiation in various spectral regions

In an attempt to detect differences in the thermal effect of infrared irradiation of different wavelengths, transient sweating response to infrared irradiation in various spectral regions was examined. In Series 1, the ventral or dorsal surface of the nude subject was irradiated repetitively for a period of 4 min (2 min on, 2 min off) by each of three kinds of infrared heaters with main emissivity in near-infrared (NIR; 0.7–2.8 m), intermediate-infrared (MIR; 1.5–5.8 m), and far-infrared (FIR; 2.8–25 m) regions. The sweating response on a non-irradiated area tended to be the greatest with MIR, while the magnitude of the sweating response on the irradiated area showed no consistent differences among various wavelengths. The results infer that MIR stimulated cutaneous thomoreceptors most effectively, while its direct effect on local sweat gland activity was minimal. In Series 2, the effects of 9–12 min irradiations in more restricted ranges of wavelength were compared by the combination of the three kinds of heaters with filters (translucent to wavelength ranges of 1.3–2.7, 2.7–3.5, 3.6–8.0 m, respectively). The sweating response on a remote area was predominantly greater with the range of 2.7–3.5 m than with the other wavelength ranges, while the local effect on sweating was minimal with this range. The results of Series 2 reinforce those of Series 1, indicating that the degree of stimulation of cutaneous thermoreceptors and of direct thermal effect on sweat gland activity differ with spectral regions incident on the skin, thus affecting local and remote effects on the sweating response.     

Nerve Growth Factor-Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells

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 PC12 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 PC12 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)].(ABSTRACT TRUNCATED AT 250 WORDS)