Association of a Notch 3 gene polymorphism with migraine susceptibility.

INTRODUCTION: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) shares common symptoms with migraine. Most CADASIL causative mutations occur in exons 3 and 4 of the Notch 3 gene. This study investigated the role of C381T (rs 3815188) and G684A (rs 1043994) single nucleotide polymorphisms (SNP) in exons 3 and 4, respectively, of the Notch 3 gene in migraine. RESULTS: The first part of the study, in a population of 275 migraineurs and 275 control individuals, found a significant association between the C381T variant and migraine, specifically in migraine without aura (MO) sufferers. The G684A variant was also found to be significantly associated with migraine, specifically in migraine with aura (MA) sufferers. A follow-up study in 300 migraineurs and 300 control individuals did not show replicated association of the C381T variant with migraineurs. However, the G684A variant was again shown to be significantly associated with migraine, specifically with MA. CONCLUSION: Further investigation of the G684A variant and the Notch 3 gene is warranted to understand their role in migraine.

[Resistance to the calcemic action of parathyroid hormone in chronic renal failure].

It is widely believed that the main mechanism of hypocalcemia in chronic renal failure is the blunted skeletal response to PTH, probably due to appearance of PTH7-84 or PTHR down-regulation in bone. We indicated that the down-regulation of renal PTHR mRNA could contribute to the development of the skeletal resistance for PTH in CRF rats. In addition, the presence of high circulating levels of non-1-84 PTH fragments (most likely 7-84 PTH) detected by the intact assay and the 1-84 PTH explain the need of high levels of intact PTH to prevent adynamic bone disease.

[Cellular mechanisms in proximal tubular handling of phosphate].

In the kidney, reabsorption of filtered inorganic phosphate (Pi) takes place along the proximal tubules and is controlled by a variety of hormones (e.g.parathyroid hormone, PTH). Three structurally unrelated sodium-dependent phosphate (Na/Pi) cotransporter families have been identified. Targeted inactivation of the type IIa Na/Pi cotransporter gene (npt2) provided strong evidence that - 70% of Na-dependent Pi transport across the brush border membrane is mediated by the type IIa Na/Pi cotransporter. The type IIa cotransporter represents the major target for PTH. The type IIa cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, PTH controlled endocytosis or the lysosomal sorting internalized type IIa cotransporter.

Secondary hyperparathyroidism and phosphate sensing in parathyroid glands.

Retention of inorganic phosphate (Pi) and associated hyperphosphatemia are important development of hyperparathyroidism secondary to renal failure. The beneficial effect of a low-Pi diet in the prevention of hyperparathyroidism can be attributed to the decrease in PTH secretion. This effect of Pi may be mediated by specific molecules in the parathyroid cell membrane. A complementary DNA encoding a Na(+)-Pi co-transporter, termed rat PiT-1, has been isolated from rat parathyroid. The amount of PiT-1 mRNA in the parathyroid is controlled by vitamin D and dietary Pi, which are the most important regulators of PTH secretion. The parathyroid Pi transporter may mediate the effects of extracellular Pi and PTH secretion in secondary hyperparathyroidism. In this study, we focus on the function of Na/Pi co-transporters in the parathyroid glands as inorganic Pi sensor.

Analysis of the fatty acid components in a perchloric acid-soluble protein.

We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Muñoz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.

Vitamin B6 suppresses growth and expression of albumin gene in a human hepatoma cell line HepG2.

The effect of vitamin B6 on the growth of a human hepatoma cell line HepG2 in culture was studied. The growth of HepG2 cells and protein synthesis were almost completely inhibited in medium supplemented with 5 mM pyridoxine. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed no inhibitory action. The growth inhibition of HepG2 cells by pyridoxine was accompanied by a marked inhibition of secretion of plasma proteins, particularly albumin. Northern blot analysis of albumin mRNA showed that pyridoxine caused a rapid decrease in the expression of albumin gene. The electron-microscopic examination of pyridoxine-treated HepG2 cells revealed a smoothing of nuclear membrane, a decrease in the number of nucleoli, and an appearance of aggregated heterochromatin structures. These morphological features are compatible with the depressed transcriptional activity in the pyridoxine-treated cells. The mechanism by which vitamin B6 exerts its inhibitory effect was discussed in terms of our recent finding that vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding proteins. Binding of pyridoxal phosphate with tissue-specific transcription factors may reduce the capacity of these factors to interact with the regulatory region of albumin gene, resulting in the inhibition of the gene expression.

Age-related occurrence of inhibitory antibodies to streptococcal pyrogenic superantigens.

Several bacteria, such as staphylococci and streptococci, can produce superantigens (SA) that induce the activation of T cells in humans. Although these organisms are the major causes of infection in children, the evidence that T cells are vigorously activated by SA produced by such organisms has not been reported except for toxic shock syndrome. In a previous paper, we demonstrated that inhibitory IgG antibodies (Ab) to SA in humans may protect against SA stimulation. In the present study, we investigated the occurrence of these inhibitory Ab to SA in 94 healthy children by the enzyme linked immunosorbent assay technique and the suppressive effect on T cell stimulation by SA. The positivity of Ab to streptococcal pyrogenic exotoxin (SPE)-A, SPE-C and staphylococcal enterotoxin B (SEB) increased with age. The age at which more than 50% of children exhibited Ab to SA was 1 year for SEB, 6 years for SPE-C and 11 years for SPE-A. Sera from these children were inhibitory to T cell proliferation elicited by SA in proportion to the concentration of IgG Ab to each SA. Sera supplemented with IgG Ab to SA by gamma-globulin therapy became inhibitory to T cell proliferation by SA. We conclude that, as children grow, they can develop Ab to SA that may play a role in protecting them against vigorous T cell activation by SA.

Vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding protein in rat liver.

The level of albumin mRNA in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of control rats. Since the transcriptional activity of the albumin gene, as measured by a nuclear run-on assay, was increased 5-fold in vitamin B6 deficiency, the higher concentration of albumin mRNA in the liver of vitamin-deficient rats could be attributed to the enhanced rate of transcription. The promoter proximal sequences of the albumin gene interact with a number of tissue-specific transcription factors including HNF-1 and C/EBP. We determined the binding activities of liver nuclear extracts to the HNF-1- and C/EBP-binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of vitamin B6-deficient rats were greater than those of controls. As the concentrations of C/EBP in nuclear extracts from control and vitamin-deficient rats, estimated by Western-blot analysis, were essentially the same, the lower binding activity of the extract from control liver is probably due to inactivation of tissue-specific factors by pyridoxal phosphate and/or its analogues. We therefore examined the effect of pyridoxal phosphate and its analogues on the binding activity of nuclear extract in vitro and found that only pyridoxal phosphate effectively inhibited the binding. These observations indicate that vitamin B6 modulates albumin gene expression through a novel mechanism that involves inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate.

Production of functional chick liver HMG 2a protein in Escherichia coli.

An efficient Escherichia coli system for the production of a variant form of high-mobility group-2a protein (HMG 2a), having the additional 5 amino acid residues (Ala-Pro-Thr-Leu-Glu) at the NH2-terminal, has been constructed. cDNA encoding HMG 2a was ligated with the Omp A signal peptide sequence and was inserted into an inducible bacterial expression vector pSH-L. After the plasmid introduced into E. coli was expressed by temperature shift, the recombinant product was purified by trichloacetic acid precipitation followed by Bio-Rex 70 column chromatography. The purified product showed the expected NH2-terminal sequence and the superhelical activity of circular DNA similar to the authentic HMG 2a isolated from chick liver.

Pyridoxal 5'-phosphate modulates expression of cytosolic aspartate aminotransferase gene by inactivation of glucocorticoid receptor.

The level of mRNA for cytosolic aspartate aminotransferase (cAST) in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of the control rats. The administration of hydrocortisone to adrenalectomized vitamin B6-deficient rats induced expression of hepatic cAST mRNA and the induction was suppressed by the simultaneous administration of pyridoxine. Since the 5' regulatory region of the rat cAST gene contains several sequences showing homology to glucocorticoid-responsive elements, we synthesized an oligonucleotide probe of glucocorticoid-responsive element sequence and assayed the binding activity of liver nuclear extract to the oligonucleotide by gel mobility shift analysis. We found that the binding activity of nuclear extract prepared from the liver of vitamin B6-deficient rats was far greater than that of the control rats, indicating that the DNA-binding activity of glucocorticoid receptor was enhanced by vitamin B6 deficiency. We further found that preincubation of the nuclear extract from the vitamin-deficient liver with pyridoxal 5'-phosphate brought about a rapid and extensive decrease in the binding of the extract to the glucocorticoid-responsive element. Congeners of pyridoxal phosphate, such as pyridoxamine 5'-phosphate, pyridoxal, pyridoxamine and pyridoxine, did not show an inhibitory effect. These observations suggest that pyridoxal 5'-phosphate modulates cAST gene expression by inactivating the binding activity of glucocorticoid receptor to glucocorticoid-responsive elements.

Effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle.

The effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle was investigated. The level of phosphorylase mRNA in the muscle of vitamin B6-deficient rats was reduced to 40% of that in the control rats. By contrast, the phosphorylase mRNA level was increased 5-fold in the liver of the deficient animals. It was also found that the expression of the beta-actin gene, generally regarded as a 'housekeeping' gene, was unaffected by B6 deficiency in the muscle but was enhanced in the liver of the deficient animals. These observations suggest that vitamin B6 may modulate the transcriptional activation of the phosphorylase gene in a tissue-specific manner.

Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver.

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6-deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.

[Quantitative analyses of the normal throat flora of children with upper respiratory tract infections].

Relationship between the normal throat flora and pathogenic bacteria recovered from the throat in 139 children with upper respiratory tract infections in winter were studied using quantitative analyses. Pathogenic bacteria examined include S. pyogenes, H. influenzae, S. aureus, and S. pneumoniae, and the normal floras include alpha-streptococci, gamma-streptococci, Neisseria species, and Micrococci. Children with S. pyogenes in their throats (S. pyogenes group) were examined with anti-streptococcal antibodies such as anti-streptolysin O, anti-streptokinase, and anti-deoxyribonuclease B. Eighty seven pathogenic bacteria were recovered from 72 children (51.8%) out of 139. S. pyogenes and S. pneumoniae groups showed significantly lower alpha-streptococci and gamma-streptococci in incidence of appearance when compared with children with the no pathogenic bacteria in their throats (no bacteria group). H. influenzae group showed significantly lower gamma-streptococci and higher Neisseria sp. in incidence of appearance compared with the no bacteria group. Positive cases for anti-streptococcal antibodies showed a significantly lower alpha-streptococci in number compared with negative cases for antibodies and the no bacteria group, and a significantly lower gamma-streptococci in incidence of appearance compared with the no bacteria group. These data suggest that the normal throat flora may have a role in prevention of colonization by the pathogenic bacteria in vivo, as were shown in vitro by many authors, and that the quantitative analysis of the normal flora is useful because this methodology might reveal whether the bacteria recovered from the throat show the pathogenicity.