An acceleration of age-related increases in levels of the beta-subunit of nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8).

An investigation was made of age-related changes in levels of the beta-subunit of nerve growth factor (beta-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of beta-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of beta-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of beta-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of beta-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.

Protection by L-ascorbic acid against phototoxicity in tin-protoporphyrin-treated suckling rats.

The protective effect of free radical scavengers against phototoxicity was investigated with tin-protoporphyrin (SnPP)-treated suckling rats. Six kinds of scavengers (L-ascorbic acid, reduced glutathione, alpha-tocopherol, retinol, uric acid and cystine) were intraperitoneally injected to rats treated with SnPP plus photoirradiation. Among them, L-ascorbic acid was found to be most effective in protecting SnPP-treated rats against phototoxicity. The survival period was markedly prolonged, and the frequency of abnormal behaviors was reduced with the treatment. Lipid peroxidation in vitro with the brain membrane fraction was also suppressed. The other five substances gave only a little antioxidant effect both in vivo and in vitro. The present study shows that L-ascorbic acid may be a promising chemical to prevent the phototoxicity of SnPP.

Cobalt-mesoporphyrin inhibits heme oxygenase activity but it does not induce lipid peroxidation in rat brain membranes during photoirradiation.

We examined the effects of cobalt-mesoporphyrin (CoMP) in vitro. The porphyrin inhibited the activity of rat splenic heme oxygenase but scarcely stimulated peroxidation of lipids in a membrane fraction from rat brain during photoirradiation. The apparent inhibition constant for CoMP was 344 nM. It is suggested that CoMP may be a promising candidate for a chemopreventive of neonatal hyperbilirubinemia that is not associated with phototoxicity.

Nerve growth factor-induced changes in the structure of sulfated proteoglycans in PC12 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)

Absence of erythrocyte arginase protein in Japanese patients with hyperargininemia.

In Japan, hyperargininemia has been reported in only 5 unrelated families and four patients are alive at present. In this study we examined arginase protein in erythrocytes of these Japanese patients using two analytical methods of immunoblotting and two-dimensional gel electrophoresis. Immunoblotting study with anti-E. coli-expressed human liver arginase rabbit IgG revealed lack of cross-reacting materials in the erythrocyte lysates from these patients. On two-dimensional gels, arginase protein was detected in any control subject, but it was completely absent in all the patients studied. These results suggest that either arginase protein in erythrocytes is not produced or it is structurally labile in these patients.

Evidence for the presence of L-arginine in the glial components of the peripheral nervous system.

L-Arginine is a precursor of nitric oxide that has been identified as an endogenous activator of soluble guanylate cyclase. We have recently reported the immunocytochemical localization of free L-arginine in glial cells in the central nervous system (CNS) using specific anti-arginine antibody. In the present study, we focused our attention on this particular amino acid in the peripheral nervous system (PNS). In the cochlea of the inner ear, arginine-like immunoreactivity was localized in satellite cells surrounding neurons of the spiral ganglion. In the dorsal root ganglia, satellite cells surrounding sensory neurons were found to be immunoreactive. In the superior cervical ganglion, L-arginine was concentrated in satellite cells around neuronal cells. In ganglia of the enteric plexus, supporting cells that covered neuronal cells were stained. These results show that free L-arginine in the PNS is concentrated in satellite and supporting cells, both of which correspond to glial cells in the CNS. Thus, those cells in ganglia of the PNS may support and/or control the neural activity by providing L-arginine to the neurons that they surround.

Elevated concentrations of beta-nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8).

Levels of the beta-subunit of nerve growth factor (beta-NGF) were determined in various tissues from senescence-accelerated mice (SAM-P/8) and compared with those from senescence-resistant control mice (SAM-R/1) at 4 months of age. (1) In SAM-P/8, the testis was 30% larger in terms of wet weight than that from SAM-R/1, whereas the adrenal glands from males and females were smaller than those from the respective controls by 45% and 20%, respectively. (2) About 70% of SAM-P/8 individuals had high concentrations of testosterone in serum (greater than 5ng/ml). (3) In SAM-P/8, endogenous levels of beta-NGF were significantly higher in the adrenal gland (20 and 7 times higher on average in males and females, respectively), in the thymus (100 and 5 times higher in males and females, respectively) and in the testis (500 times higher) than those in the control tissues. In other tissues there were little or no differences in terms of levels of beta-NGF. (4) Morphological changes in the adrenal gland, thymus and testis of SAM-P/8 mice were not as marked as expected from the elevated levels of beta-NGF in these tissues. (5) These results show that, in SAM-P/8 mice at 4 months of age, an elevation in the endogenous level of beta-NGF has already occurred in some peripheral tissues before senescence becomes accelerated.

Genetic defect of bilirubin UDP-glucuronosyltransferase in the hyperbilirubinemic Gunn rat.

The genetic defect of bilirubin UDP-glucuronosyltransferase (UDPGT) in the hyperbilirubinemic Gunn rat was proved to be a -1 frameshift mutation. The mutation was found not only to be located in the region where bilirubin UDPGT cDNA shared an identical sequence with 3-methylcholanthrene (3M C)-inducible UDPGT cDNA but also to occur in the same position on the two cDNAs from the mutant rat. At the 5' end of the identical region there was a consensus sequence for splicing, of which position coincided with the boundary between the 2nd and 3rd exon of the testosterone UDPGT gene. These results strongly suggest that mRNAs for bilirubin and 3M C-inducible UDPGTs are produced from a single primary-transcript after an alternative splicing and the defects of bilirubin and 3M C-inducible UDPGTs in the mutant rat are caused by a point mutation on a common exon.

Thyroid hormone metabolism and nuclear binding in Gunn rats.

We examined the serum concentrations of total, free thyroid hormones and TSH, activity of hepatic T4 5'-deiodinase, and T3 binding to hepatic nuclei in homozygous (j/j) and heterozygous (j/+) Gunn rats. Both total T3 and free T3 (FT3) concentrations in sera from j/j rats were significantly lower than those of j/+ rats on 5-10, 15-20, and 25-30 days after birth. Both total T4 and free T4 (FT4) concentrations in j/j and j/+ rat sera were not significantly different on 5-10 days. However, in j/j rats they were significantly higher than those of j/+ rats on days 15-20 and 25-30. Serum reverse T3 (rT3) concentrations were higher in j/j than in j/+ rats on days 5-10, 15-20, and 25-30. Serum TSH concentration in j/j and j/+ rats on 15 days post-natal were 1.42 +/- 1.28 and 1.65 +/- 1.24 micrograms/l (mean +/- SD), respectively, which were not significantly different from each other. T3 formation from T4 in hepatic microsomal fractions obtained 15 days after birth was significantly lower in homozygotes than in heterozygotes (4.89 +/- 1.18 vs 11.15 +/- 2.38 pmol/mg protein/min, p less than 0.005). Binding constants (Ka) as well as maximal binding capacities (MBC) for T3 of hepatic nuclei from 15 day-old j/j and j/+ rats were similar (ka; 3.58 x 10(9) vs 3.15 x 10(9) M-1, MBC; 0.316 vs 0.380 pmol/mg DNA). From these results we suggest that decreased conversion from T4 to T3 is one of the major reasons for high serum levels of T4 and rT3, and low levels of T3 in j/j rats, and that nuclear T3 binding and pituitary TSH secretion are unaltered in j/j rats.

Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence.

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group of L-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of L-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specific anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with L-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of L-arginine, a nitric oxide precursor, in the brain.

Sn-protoporphyrin plus photoirradiation induces lipid peroxidation in vivo and in vitro in nonjaundiced Gunn rats.

Lipid peroxidation induced by Sn-protoporphyrin (SnPP) plus photoirradiation was investigated in vivo and in vitro using nonjaundiced Gunn rats. Membrane lipids from young adult rat brain were peroxidized by SnPP plus photoirradiation depending on the SnPP concentration and photoirradiance. Similarly, coadministration of SnPP and photoirradiation to suckling rats increased lipid peroxides in the whole blood and was found lethal. The influence of the wavelength distribution of light sources was also examined by using blue-white and green fluorescent lights. The photodynamic effect by green light irradiation whose energy distribution had no overlap with the Soret band of SnPP was about half of that produced by blue-white light with regard to the membrane peroxidation and the lethal effect on neonatal rats. We therefore conclude that the combination of SnPP and photoirradiation is a potentially hazardous treatment of neonatal jaundice.

Distribution of acid phosphatase and beta-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats. An enzyme histochemical study.

Activities of acid phosphatase and beta-glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although beta-glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.

Changes in glycosaminoglycans during the neuritogenesis in PC12 pheochromocytoma cells induced by nerve growth factor.

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 approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Coexistence of parvalbumin and glycine in the rat brainstem.

The coexistence of glycine- and PV-immunoreactivities was studied immunocytochemically in the nuclei of the superior olive, trapezoid body, cochlea and lateral lemniscus. All of the PV-immunoreactive neurons in the nuclei of the superior olive and trapezoid body were immunoreactive to glycine but not to GABA. In the dorsal cochlear nucleus, PV-positive neurons were sometimes immunoreactive to glycine. In the ventral nucleus of the lateral lemniscus, PV-positive cells were immunoreactive neither to glycine nor to GABA. Consequently, it was concluded that PV-immunoreactivity was distributed not only in the GABAergic neurons, but also in the glycinergic neurons and possibly in wider neuronal populations.

Influences of neonatal and adult exposures to testosterone on the levels of the beta-subunit of nerve growth factor in the neural tissues of mice.

In our previous report, we have shown the sex difference in the concentration of the beta-subunit of nerve growth factor (beta-NGF) in the neural and paraneural tissues of mice. In this investigation, we examined the effects of castration of adult males, and of neonatal and/or adult treatments with testosterone on levels of beta-NGF in the several tissues of mice. Castration caused a marked reduction in the levels of serum testosterone and of beta-NGF in the brain, spinal cord and submandibular glands, but not in the pancreas and kidneys. Continuous infusion of testosterone for one week into adult males that had been castrated at 2 months of age restored the level of beta-NGF in the three tissues mentioned above. A single injection of testosterone to 5-day-old female pups to masculinize the brain gave no effect on the level of beta-NGF in any tissue dissected after 4 months. A one-week infusion of testosterone into adult females slightly increased levels of beta-NGF in the brain and spinal cord, but the same treatment of adult females given in advance a single dose of testosterone at 5 days of age caused a significant increase in its levels over those of untreated females. These results suggest that neonatal and adult exposures to testosterone can influence the endogenous concentration of beta-NGF in the brain and spinal cord.

Isolation and sequencing of rat liver bilirubin UDP-glucuronosyltransferase cDNA: possible alternate splicing of a common primary transcript.

A 1763-bp cDNA for rat liver bilirubin UDP-glucuronosyltransferase (UDPGT) was isolated. Bilirubin UDPGT activity was demonstrated by transfection of the pcDL1 vector carrying the cDNA into COS7 monkey kidney cells. The cDNA shares an identical 913-bp sequence (corresponding to the C-terminal 247 amino acid residues) with that for rat liver 3-methylcholanthrene-inducible 4-nitrophenol UDPGT including the locus where a -1 frameshift mutation was found in the 4-nitrophenol UDPGT cDNA from the jaundiced homozygous Gunn rat. The result suggests that both the UDPGTs are derived from a common primary-transcript and that the multiple defects of UDPGT isoenzymes observed in the homozygous Gunn rat may be produced by a single-mutated-locus after an alternative splicing of the 5' end region.

Dangerous effects of tin-protoporphyrin plus photoirradiation on neonatal rats.

In vivo and in vitro effects of porphyrins (tin-protoporphyrin [SnPP], cobalt-mesoporphyrin, haemin and protoporphyrin) on neonatal rats were investigated. Under photoirradiation a high mortality rate was recognized in SnPP injected rats. None died from the application of SnPP without photoirradiation. In photoirradiated rats the median lethal dose (LD50) value of SnPP was calculated to be about 7.4 mumol/kg body weight. Haemolysis and malonaldehyde formation of red blood cells were induced by SnPP together with photoirradiation. SnPP may be useful in reducing bilirubin levels in severely jaundiced infants under non-photoirradiated conditions or dim light, but prophylactic administration of SnPP to the majority of infants is not recommended.

When does GABA-like immunoreactivity appear in the rat cerebellar GABAergic neurons?

The time of appearance of gamma-aminobutyric acid (GABA), a well-known neurotransmitter, during the development of cerebellar GABAergic neurons in rats was investigated immunocytochemically using purified anti-GABA antibody. Sprague-Dawley rats were used at embryonic days 15, 16, 18, 19 and 21, and postnatal days 0, 1, 5, 10, 15, 20 and 30. Golgi cells showed processes and GABA-like immunoreactivity at embryonic day 16 during migration. Purkinje cells were found immunoreactive at embryonic day 18, when they arrived at their destination. The reactivity of the basket cell was already apparent at postnatal day 5, and was thought to appear just after the end of migration. In all of the GABAergic neurons, GABA-like immunoreactivity was visible much earlier than the time of synapse formation and the emergence of their electrophysiological activity described in the literature. In addition, GABA-like immunoreactivity tended to shift from the soma and dendrite into the axon with development.