Biomedical subjects
Publications and source records attributed to S Furukawa.
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We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
Intraperitoneal administration of kainic acid (KA) into adult rats caused a profound increase in nerve growth factor (NGF) mRNA and a significant reduction of NGF protein level in the hippocampus. Diazepam pretreatment suppressed both. The reduction of NGF level was apparent in the dorsal hippocampus at 2 h after KA administration, but a marked elevation of NGF protein was observed in the ventral hippocampus at 4 h. These results suggest that non-N-methyl-D-aspartate (NMDA) receptor agonists negatively influence NGF synthesis or stimulate NGF protein degradation in the dorsal hippocampus involving the CA1 sector.
Involvement of nerve growth factor (NGF) in the pathogenesis of delayed neuronal death (DND) of CA1 neurons in the hippocampus has been suggested. We measured regional changes in the content of tissue NGF of the hippocampus in the Mongolian gerbil after 5 min forebrain ischemia. The NGF content was found to decrease significantly in the CA3 and dentate regions by 32% two days after ischemia. By contrast in the CA1 region, the level of NGF became significantly elevated by 50% two weeks after ischemia or later. The early reduction of NGF content in the afferent area projecting to the CA1 sector might be primarily linked to the pathogenesis of DND, whereas the delayed increase within the CA1 sector might be a secondary local response mainly of reactive astroglia.
The results of radiotherapeutic treatment in 71 patients with squamous cell carcinoma of the cheek mucosa were reviewed. The actuarial 5-year local control rate was 100% for T1 (8 patients), 62% for T2 (43), 65% for T3 (17) and 0% for T4 (3). The patients were divided into four groups according to treatment modality; group 1 was treated by radiotherapy alone (R), group 2 by radiotherapy combined with chemotherapy of BLM or PEP (R + C), group 3 by external radiotherapy followed by surgery (R + S) and group 4 by a combination of radiotherapy, chemotherapy and surgery (R + C + S). The 5-year local control rate was 44% for R (11 patients), 61% for R + C (39), 63% for R + S (6) and 80% for R + C + S (15). Nine of 14 cases or 64% of the surgical specimens in the R + C + S group showed no tumor cells microscopically, a rate comparable with the 5-year local control rate of the R + C group. Including the results of secondary treatment by surgery for recurrent cases, the ultimate local control rate was 83% in both the R and R + C groups. The local control rate was 88% for carcinoma located in the anterior half of the cheek and 53% for that in the posterior cheek. The results suggested that tumors extending to the bucco-alveolar sulci would be more difficult to control by radiotherapy alone, with or without chemotherapy.
Acidic fibroblast growth factor (aFGF) level in sciatic nerve after lesioning was measured by enzyme immunoassay to determine if aFGF functions as a neurotrophic factor like nerve growth factor (NGF). Whereas the NGF level increased in distal segments, the aFGF level there decreased after transection or crushing and recovered to the original level by 10 weeks after crushing. The amount of aFGF mRNA in the sciatic nerve was extremely low to supply the high level of protein found in the sciatic nerve. Sympathetic ganglia, dorsal root ganglia, and spinal cord, which contain neuronal cell bodies extending their axons into the sciatic nerve, showed a greater or similar level of aFGF as sciatic nerve. These results imply that aFGF is synthesized in neuronal cell bodies and distributed anterogradely into their axons. Difference of injury-induced changes in levels between aFGF and NGF suggests distinct mechanisms of the effects elicited from these factors on regeneration of the sciatic nerve.
OBJECTIVE: We investigated whether levels of intercellular adhesion molecule 1 (ICAM-1) antigen shed into the circulation increase during acute Kawasaki disease (KD). We also compared ICAM-1 levels in acute KD with those in anaphylactoid purpura (AP) and in measles. METHODS: Serum ICAM-1 levels were measured by a double-determinant immunoassay using 2 monoclonal antibodies in the FAST (Falcon assay screening test) system. Serum levels of tumor necrosis factor alpha (TNF alpha) were measured by a specific and sensitive sandwich enzyme immunoassay. RESULTS: Patients with KD, but not those with AP or measles, had increased levels of shed ICAM-1 antigen in serum samples obtained during acute stages. Moreover, during the acute stage, KD patients with coronary artery lesions (CAL) had still higher levels of shed ICAM-1 than did those without CAL. We found a positive correlation between serum levels of shed ICAM-1 and levels of TNF alpha during acute KD. CONCLUSION: Our findings suggest that the serum ICAM-1 level is an important immunologic parameter for determining the severity of vascular damage during acute KD.
In serum, nerve growth factor (NGF) forms a complex with alpha 2-macroglobulin (alpha 2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF-alpha 2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system.
It has been reported that tumour necrosis factor-alpha (TNF-alpha) is capable of inducing vascular injury, and interleukin 6 (IL-6) of inducing production of acute phase proteins and the maturation of megakaryocytes. Kawasaki disease (KD) is a systemic vasculitis with severe inflammation. We investigated whether TNF-alpha and IL-6 activities in serum from patients with KD differs from those in anaphylactoid purpura (AP) and measles. Serum TNF-alpha levels were measured by a sandwich enzyme immunoassay and IL-6 activities in serum were assessed by a colorimetric assay. Both KD and AP patients but not patients with measles had increased serum TNF-alpha levels during the acute stage. With respect to IL-6, patients with KD and measles, but not AP, had increased IL-6 activities in serum during the acute stage. IL-6 activities in serum of KD patients correlated with serum C-reactive protein levels and correlated to some extent with maximum platelet counts during the course of illness. These results suggest that KD differs from AP and measles regarding both cytokines. The combination of TNF-alpha, which may be responsible for severe vascular injury, and IL-6, which may be responsible for severe inflammation, may play an important role in acute KD.
This study examined the effects of 4-methylcatechol (4-MC), a nonamine catechol compound, on the neuropathic process of streptozotocin (STZ)-induced diabetic rats. 4-MC is one of the potent stimulators of nerve growth factor (NGF) synthesis at the cellular level and in cultured sciatic nerve segments of rats. Diabetic rats showed a statistically significant fall in sciatic motor nerve conduction velocity (MNCV) and a significantly reduced NGF content in the sciatic nerve (38.5 +/- 2.8% of control, P less than 0.01) during the experimental period of 4 weeks. 4-MC treatment of the diabetic rats for 4 weeks starting from the STZ injection elevated the NGF content (140% of untreated diabetic rats, P less than 0.05) and prevented the reduction in MNCV, but no effect on high blood glucose levels was seen. These findings suggest that decreased NGF levels in the sciatic nerve of the experimental diabetic rat may be involved in the development of the diabetic neuropathic process and that 4-MC, which can elevate endogenous NGF levels in vivo, may compensate for the inhibitory effect of STZ on the NGF level in progressive diabetic neuropathy.
We modified a previously reported enzyme immunoassay method to make it more sensitive for quantification of nerve growth factor (NGF), and succeeded in measuring the NGF content in as small as 2 mg (wet weight) of rat brain tissue. Rat brain was cut into about 600 pieces of the same size, and the NGF content in each piece was determined by this method. The findings were as follows: (i) In the cerebral cortex, NGF contents were unevenly distributed, ranging from less than 0.1 to 1.8 ng/g wet wt. The level was highest in the caudal parietal and rostral occipital cortices and lowest in the lateral parietal cortex. (ii) Areas comprising the limbic system such as the cingulate gyrus, pyriform cortex, amygdala, anterior and medial thalamus, hippocampus, septum, and diagonal band of Broca contained high levels of NGF. (iii) In the brain stem and cerebellum, the levels were low; however, a relatively high level was registered in the cerebellar nuclei, lateral vestibular nucleus, ventral cochlear nucleus, superior olive, and pontine reticular nuclei. These findings, taken together with previously published information, suggest that the neurons in the anterior and medial thalamus, pontine reticular nuclei, superior olive, ventral cochlear nucleus, and cerebellar Purkinje cells may be additional populations of NGF-responsive neurons in the rat brain.
Very-low-density lipoprotein (VLDL) triglyceride turnover was examined in mildly streptozotocin (25 mg/kg)-diabetic rats, using Triton WR1339. Diabetic rats fed standard rat chow showed mild hyperglycemia and suppressed levels of plasma insulin. Their triglyceride secretion was significantly suppressed despite an elevated level of plasma free fatty acids. However, the plasma triglyceride level of these diabetic rats was significantly elevated compared with nondiabetic controls. This suggested that the removal of triglyceride from the circulation, as well as its entry into the circulation, was impaired in mildly insulin-deficient rats. Glucose or fructose supplementation (10% in drinking water for 14 days) significantly increased the triglyceride secretion rate of diabetic rats. Especially, fructose supplementation increased plasma insulin to normal levels, but resulted in markedly elevated plasma triglyceride levels (three times higher than glucose-supplemented or chow-fed diabetic rats) despite similar triglyceride secretion rates between the two types of sugar-supplemented diabetic rat groups. This suggested an impairment of triglyceride removal by dietary fructose. The result obtained from chow-fed diabetic rats indicates that mild but significant insulin deficiency resulted in mild hypertriglyceridemia, linked to impaired triglyceride removal rather than to an overproduction of VLDL-triglyceride, despite elevated levels of plasma free fatty acids. Furthermore, fructose feeding induced prominent hypertriglyceridemia not only by stimulating triglyceride secretion, but also by suppressing triglyceride removal from the circulation of mildly diabetic rats.
Recently it has been reported that naturally occurring inhibitors of tumor necrosis factor alpha (TNF-alpha) were demonstrated in urine of some acute febrile patients. We investigated whether TNF-alpha inhibitory activity in urine increases during acute Kawasaki disease (KD). TNF-alpha inhibitory activities in urine were measured by a cytotoxicity assay on the TNF-susceptible cell line L929. KD patients had increased TNF-alpha inhibitory activities in urine during the acute stage and returned to a normal range during the convalescent stage. Our results suggest that the TNF-alpha inhibitor in urine is part of the regulatory system of TNF-alpha, which might be responsible for vascular injury during acute KD.
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OBJECTIVE: Increased sympathetic innervation has been reported in spontaneously hypertensive rats (SHR); however, the precise mechanisms involved are not yet clear. Nerve growth factor (NGF), a neurotrophic peptide in peripheral sympathetic neurons, is believed to contribute to this phenomenon. METHODS: We measured the content of NGF in SHR and control Wistar-Kyoto (WKY) rats during development. Mesenteric artery, spleen, heart and sciatic nerve were isolated and homogenized. NGF content in the supernatant fractions was measured using a highly sensitive and specific two-site enzyme immunoassay. RESULTS: At 3 weeks of age, SHR had a greater NGF content in the spleen, the sciatic nerve and the mesenteric artery than WKY rats. However, these differences disappeared completely at 12 weeks of age. Cardiac NGF content was slightly lower in 3-week-old SHR and, conversely, higher in 12-week-old SHR than in age-matched WKY rats. CONCLUSIONS: These findings suggest that, except for the heart, the SHR tissues observed overproduce NGF at a young age, leading to enhancement of peripheral sympathetic nervous system activity and the production of vasoconstrictive catecholamines.
IgE levels in faecal extracts (Copro-IgE levels) were investigated in food allergy (FA) patients before and after the challenge test administration of food allergens. IgE levels were measured by time-resolved fluoroimmunometric assay. In addition, the effects of administration of oral sodium cromoglycate (SCG) on the Copro-IgE levels were studied. Copro-IgE levels in patients with FA, who were placed on an elimination diet, did not differ from those of healthy children. After a challenge test immediate symptoms of urticaria and wheezing were observed in all FA patients. Copro-IgE levels in each patient increased markedly within 24 h of the challenge test. Moreover, FA patients treated orally with SCG showed neither the increase in Copro-IgE levels nor any remarkable symptoms after the challenge. Our results suggest that the increased Copro-IgE levels may be a specific consequence of the local immune response to food allergen stimulation in the gut mucosa.
The peripheral blood mononuclear cell subsets in patients with Kawasaki disease and coronary artery lesions were investigated. Of the 106 patients 14 had lesions. Patients with Kawasaki disease and coronary artery lesions were found to have increased counts of CD14+ macrophages/monocytes compared with those of patients with Kawasaki disease without lesions. The absolute counts of CD14+ macrophages/monocytes form an important parameter to determine the severity of vascular damage during acute Kawasaki disease.