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A simple spectrophotometric assay of phosphoribosyl pyrophosphate synthetase from human erythrocytes is described. The kinetic properties of the enzyme have been determined and are in good agreement with published results. Conditions for storing haemolysates at -20 degrees C are described. The method is suitable for semi-automation.
Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 mumol/L, easy to perform and gives results comparable to more complex methods. Daily urinary output of PPi was independently related to both age (P = 0.0014) and sex (P = 0.0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P = 0.012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume (P = 0.004) and with phosphate excretion (P = 0.008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.
Calcium and fluoride release from excess solid calcium fluoride was monitored for 15-30 min in aqueous solutions containing various concentrations of inorganic orthophosphate and pyrophosphate. Low concentrations of these ions (1-10 mumol/L) considerably inhibited the rate of dissolution of calcium fluoride. This inhibition was pH-dependent, being reduced at pH values below 5. It is suggested that a reduced calcium fluoride dissolution rate, in the presence of phosphate, can account for the relatively slow loss of calcium fluoride from dental enamel observed in recent clinical studies. It also appears that calcium fluoride coated with phosphate may provide a pH-controlled slow release of fluoride that may be of clinical significance and a major component of the cariostatic mechanism of topically applied fluoride.
Gammadelta T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether gammadelta T-lymphocytes produce fibroblast growth factor (FGF)-9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood gammadelta T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor-beta1 (TGF-beta1)/interleukin-15 (IL-15) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human gammadelta T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-beta1/IL-15 did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon-gamma mRNA. Western blot analysis of gammadelta T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage gammadelta T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.
OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) deposition disease (CPPDD), also known as pseudogout, is rarely known to affect the spine. The purpose of this article is to report our experience with six cases involving massive focal deposition of CPPD crystals in the ligamentum flavum. METHODS: Between January 1998 and June 2002, we treated six patients with CPPDD involving the ligamentum flavum of the cervical and thoracic spine. Their ages ranged from 45 to 70 years. There were five female patients and one male patient. The cervical spine was involved in two cases and the thoracic spine in four. All except one patient presented with an insidious onset of myelopathy. The remaining patient presented with paraplegia after trauma. None of the patients exhibited any systemic features of CPPDD or other metabolic conditions that can lead to CPPD deposition. Plain x-rays often yielded inconclusive results. Computed tomography and magnetic resonance imaging were useful in confirming the diagnoses. Decompressive laminectomy, with removal of the ossified ligamenta flava, was performed for all patients. Polarized-light microscopic examinations of the excised ligamenta flava revealed the characteristic rod-shaped, birefringent crystals. RESULTS: Five of the six patients experienced significant improvements in their myelopathic symptoms after surgery. The remaining patient experienced improvements in sensations but no appreciable improvement in motor power. During the follow-up periods, which ranged from 7 months to 3 years, none of the patients presented with a recurrence of CPPD crystal deposition at the previously treated level. However, one patient who exhibited improvement after surgery presented 2 years later with a recurrence of myelopathic features attributable to ossification of the ligamentum flavum at a new level. CONCLUSION: Tumoral CPPDD of the ligamentum flavum is rare. It commonly occurs among middle-age or elderly female patients and presents with progressive myelopathy. Computed tomography and magnetic resonance imaging are complementary in the diagnosis of this condition. Surgery, if performed early, leads to good improvement. However, long-term follow-up monitoring of these patients is necessary, because surgery provides only symptomatic relief and does not treat the underlying disease. With the increasing availability of magnetic resonance imaging, ossification of the ligamentum flavum is being more frequently recognized. In every case of ossified ligamentum flavum, the excised specimen should be examined with polarized-light microscopy. We think that this simple, inexpensive method will lead to the recognition of more cases of spinal CPPDD.
The 45- and 35-kDa subunits of mevalonate pyrophosphate decarboxylase (MPD) have been purified from rat liver. In this study, we examined the relationship between 45- and 35-kDa MPD and the tissue distribution of a major MPD in rat liver. When the crude extract of rat liver fed on normal chow was subjected to immunoblot analysis using anti-rat 45-kDa MPD antibody, only the 45-kDa band was detected. In a pulse-chase experiment using anti-rat 45-kDa MPD antibody, there was no precursor-product relationship between the 45- and the 35-kDa MPD. In immunoprecipitation, more than 85% of MPD activity in the rat liver was depleted from the crude extract with an excess of the above antibody. When 45-kDa MPD contents in tissues were analyzed by immunoblotting, a single protein band with an apparent molecular weight of 45 kDa was detected in all tissues. The specific protein content of 45-kDa MPD in liver was markedly higher than in other tissues. The activity/amount ratio varied among brain, liver, and testis, being significantly highest in the liver. From these data, it is suggested that 45-kDa MPD serves as a major enzyme involved in cholesterol biosynthesis in rat liver and that a tissue-specific regulator or isozyme of 45-kDa MPD is present in rat liver.
Mevalonate pyrophosphate decarboxylase (MPD) is found in the 100000 x g supernatant fraction of cells or tissues and is considered to be a cytosolic protein. Recently, other groups reported that MPD is mostly located in the peroxisomes. In this study, we used two different methods to determine whether MPD is predominantly located in the peroxisomes or the cytosol of rat hepatocytes. 1) In permeabilized rat hepatocytes or normal rat kidney cells treated with digitonin, which lack cytosolic enzyme, MPD was mainly present in the medium. 2) Double immunofluorescent labeling of cells with both anti-MPD antibody and anti-hexokinase antibody yielded an immunofluorescent pattern for both enzymes typical of the cytosolic protein. These results indicate that MPD is predominantly located in the cytosol of rat hepatocytes.
We previously reported that the CP diet (a diet containing 5% cholestyramine and 0.1% pravastatin)-induced new species of 37-kDa mevalonate pyrophosphate decarboxylase (MPD) was characteristically and immunologically very similar to the well-known 45-kDa MPD. In the present study, we found a difference in subcellular distribution between 45- and 37-kDa MPD by cell fractionation and immunoblot analysis. The cytosol fraction contained 45- and 37-kDa MPD. Peroxisomal fraction contained a small amount of 45-kDa MPD, but not 37-kDa MPD. Also, 45-kDa MPD in peroxisome is localized in the matrix. From these data, the difference in subcellular distribution between 45- and 37-kDa MPD may be due to differences in the physiological role of cholesterol biosynthesis in rat liver.
We carried out a comparison of tissue distribution of mevalonate pyrophosphate decarboxylase (MPD) between normotensive Wistar Kyoto rat (WKY) and stroke-prone spontaneously hypertensive rat (SHRSP) using Western blotting. However, there was no difference in tissue distribution of MPD between WKY and SHRSP, expect in brain and liver. We then compared the MPD between WKY and SHRSP liver at several weeks of age. We found that MPD in the liver as well as brain of SHRSP was significantly reduced from two weeks of age. This data is useful to identify or understand the mechanism underlying the reduced amount of MPD in SHRSP.
We previously reported that the lower activity of mevalonate pyrophosphate decarboxylase (MPD) was caused by the reduced amount of this enzyme in stroke-prone spontaneously hypertensive rat (SHRSP) by immunoblot analysis using 20,000 x g supernatant containing cytosol and microsomes. A recent study showed that at least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. In this study, we examined the subcellular distribution of 45- and 37-kDa MPD in the liver of SHRSP and compared normotensive Wistar Kyoto rat (WKY) and SHRSP. 45-kDa MPD was detected in the cytosol and peroxisomes of SHRSP, while 37-kDa MPD was detected in the cytosol of SHRSP, but not in the peroxisomes. The relative enrichment of 45-kDa MPD in peroxisomes was lower than that of LDH, suggesting the possibility that 45-kDa MPD of SHRSP did not exist in the peroxisomes. Also, 45-kDa MPD was decreased in the crude extract containing 1% Triton X-100, cytosol and peroxisomes of SHRSP, and 37-kDa MPD was decreased in the crude extract containing 1% Triton X-100 and cytosol of SHRSP, as compared with WKY. These data indicate that the cholesterol synthesis in the liver of SHRSP by the reduced amount of MPD is significantly reduced.
We previously reported that treatment of rats with a diet containing 0.1% pravastatin and 5% cholestyramine markedly increased mevalonate pyrophosphate decarboxylase (MPD) activity in liver crude extracts compared with nontreated rats. In this study, we examined the change in the protein level of MPD in the tissues of mice administered pravastatin. When MPD content in the tissues of nontreated mice was analyzed by quantitative immunoblotting, a single protein band with an apparent molecular weight of 46 kDa was detected in all tissues and the specific protein content of MPD in liver and kidney was markedly higher than that in other tissues. When MPD content in the tissues of pravastatin-treated mice was analyzed by immunoblotting, MPD was markedly increased (9-fold) only in the liver compared with nontreated mice. Next, when MPD activity was measured in the liver between nontreated and pravastatin-treated mice, MPD activity as well as protein levels were markedly increased (11-fold) in the liver of pravastatin-treated mice compared with nontreated mice. These data suggest that a marked induction of MPD in the liver by pravastatin is responsible for the tissue-specific effect of pravastatin.
It is known that cholesterol biosynthesis in the liver is inhibited by probucol. This inhibition by probucol is caused at least in part by a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. In this study, we examined serum cholesterol and the change in the activity or protein level of mevalonate pyrophosphate decarboxylase (MPD), which is involved in cholesterol biosynthesis, in the livers of rats fed probucol. The results indicated that serum cholesterol, MPD activity and MPD protein were decreased by 70, 50 and 60% by probucol, respectively, as compared with those in rats fed normal chow. These data show for the first time that probucol decreases the level of an enzyme involved in cholesterol biosynthesis other than HMG-CoA reductase.
Mevalonate pyrophosphate decarboxylase (MPD) is considered to be a cytosolic protein. Recently, other groups reported that MPD is mostly located in the peroxisomes. In this study, we examined whether the expression of MPD in mice depends on the proliferation of peroxisomes, and whether MPD is predominantly located in the peroxisomes or the cytosol of mice. No increase in the protein level of MPD was observed in the crude extract of the livers of mice administered with peroxisome proliferative drugs. The result suggests that the expression of MPD is independent of the proliferation of peroxisomes, and may be maintained via a specific regulatory mechanism, different from the regulation of the expression of peroxisome proliferator-activated receptor alpha. When the subcellular distribution of MPD in mouse melanoma (B16F10) cells was examined by cell fractionation, MPD was detected in the cytosol of B16F10 cells, but not in the peroxisomes. In permeabilized B16F10 cells treated with digitonin, which lack cytosolic enzymes, 80% and 20% of MPD, 75% and 25% of lactate dehydrogenase, or 2% and 98% of catalase, existed in the medium and in the cell, respectively. From these results, it indicated that MPD was predominantly located in the cytosol and did not exist in the peroxisomes of B16F10 cells.
To determine whether or not the expression of mevalonate pyrophosphate decarboxylase (MPD) depends on the proliferation of peroxisomes, we examined change in the protein level of MPD in the crude extract, the cytosol and the peroxisome-enriched fraction of the livers of rats administered peroxisome proliferative drugs. No increase of MPD was observed in any of these fractions. These data suggest that the expression of MPD is independent of the proliferation of peroxisomes and may be maintained via a specific regulatory mechanism different from that of the expression of peroxisome proliferator-activated receptor alpha.
The spontaneously hypertensive rat (stroke-prone) (SHRSP) experiences severe hypertension and cerebral hemorrhage. The serum cholesterol level in this rat is lower than that in the normotensive Wistar-Kyoto rat. Epidemiologic studies have indicated a negative association between serum cholesterol level and the incidence of cerebral hemorrhage in humans. Therefore the low level of serum cholesterol in SHRSP may cause cerebral strokes. The following investigation demonstrated that the activity for the biosynthesis of cholesterol is decreased in SHRSP due to the reduced activity of mevalonate pyrophosphate decarboxylase (MPD). However, the mechanism underlying the reduced activity of this enzyme remains unclear. In this review, we indicate that the level of MPD in the brain and liver of SHRSP is reduced from the age of 2 weeks.
The myocardial uptake of technetium-99m pyrophosphate (99mTc-PYP) in perimyocarditis induced by coxsackievirus B3 in BALB/c mice was studied on 3rd--90th day after the inoculation. 99mTc-PYP uptake ratio, measured by the ratio of cpm/gm for the heart to cpm/gm for the skull, began to increase five days after virus inoculation and reached a maximum on the seventh day. After the 14th day, 99mTc-PYP ratio began to decrease, however, on the 90th day, a high 99mTc-PYP uptake was shown in mice with severe perimyocardial fibrosis and calcification. The present findings may provide a futher basis upon which 99mTc-PYP imaging may be applied to viral perimyocarditis in humans.
From Escherichia coli cells with a deletion in the ssrA gene that encodes 10Sa RNA after treatment with a mutagen, we isolated two temperature-sensitive mutants, which we designated TS15 and TS101. The temperature-sensitive (ts) phenotype of the mutants could be overcome by introduction of the wild-type ssrA gene but not by the mutants of ssrA. By a complementation test using Kohara's mini-set of clones and by subcloning of a fragment from the phage clone 246, we found that both mutations were in the prs gene that encodes phosphoribosyl pyrophosphate synthetase. Sequencing of the mutant prs gene of TS101 showed that residues 215, cysteine, in the encoded protein had been changed to tyrosine. That such a mutant exists suggests that 10Sa RNA associate with the prs gene product in a functional way.
An unnatural alpha-D-mannopyranose-linked chitobiosyl dolichyl pyrophosphate, a stereoisomer of the N-glycan biosynthesis intermediate, was synthesized. The protected trisaccharide, alpha-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-D-GlcNAc, carrying a 4-methylbenzoyl group was prepared for the convenience of a TLC analysis. 1-O-Phosphorylation, condensation with dolichyl phosphate, and subsequent deprotection afforded the title compound.