Keishi Hata

The Interaction between Ku Antigen and REF1 Protein Mediates Negative Gene Regulation by Extracellular Calcium

Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca. The Ku antigen mediated negative gene regulation by Ca by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

Sulfidoleukotriene Release Test (CAST) in Hypersensitivity to Nonsteroidal Anti-Inflammatory Drugs

There is a great need to develop a method for making an accurate and reliable in vitro diagnosis of adverse hypersensitivity reactions to drugs. We measured the amount of sulfidoleukotriene (sLT) released from the peripheral blood leukocytes obtained from 25 patients who developed hypersensitivity reactions following the administration of nonsteroidal anti-inflammatory drugs (NSAIDs); 12 patients demonstrated reactions to Voltaren, 8 patients to Bufferin, and 5 patients to Sedes G. The stimulation index, the ratio of the amounts of sLT (pg/ml) incubated with and without drugs, was considerably higher in the patients than in the controls, which consisted of 5 nonallergic healthy subjects. The sensitivity of the CAST (cellular antigen stimulation test) was evaluated to range from 62.5 to 80%, while the specificity was 70-100%. The CAST may thus be useful as a novel in vitro test system in order to screen for possible hypersensitive reactions to NSAIDs with both reliability and safety.

Differences between the Sugar Moieties of Liver- and Bone-Type Alkaline Phosphatases: A Re-Evaluation

We re-evaluated the differences between the sugar moieties of liver and bone alkaline phosphatases (ALPs). Sialic acid was added to ALP sugar moieties by α2,3- or 2,6-sialyltransferase treatment of the asialo-form ALP (neuraminidase-treated ALP). Asialo-bone ALP was converted to a liver-like ALP by the 2,6-sialyltransferase treatment. The resulting liver-like ALP was less susceptible to neuraminidase than non-treated bone ALP, but was still labile to heat exposure at 56°C like non-treated bone ALP. However, after the O-linked sugar moiety had been released by additional treatment with O-glycanase the liver-like ALP became more heat stable at 56°C, like non-treated liver ALP. Non-treated liver ALP reacted specifically with anti-liver ALP monoclonal antibody, and non-treated bone ALP reacted with both anti-liver and anti-bone ALP antibodies. The asialo-bone ALP still reacted with anti-bone ALP antibody, whereas the asialo-form liver ALP showed little, if any, reaction with anti-liver and anti-bone ALP antibodies. Neuraminidase and O-glycanase-treated bone ALP reacted less with anti-bone ALP antibody. After O-glycanase treatment, bone ALP molecules deprived of an O-linked sugar moiety had a molecular size and heat stability similar to liver ALP. The difference between liver and bone ALP molecules may be due not only to their manner of sialic acid linkage but also to the attachment of the O-linked sugar moiety.

Urinary excretion of pyridinium cross-links of collagen in infancy☆

This cross-sectional study evaluated urinary excretion of pyridinium cross-links of collagen, specific markers of ongoing bone resorption, in infants aged 1 week to 7 months and examined the relationship between urinary cross-links and individual renal function. Spot urines from a total of 100 infants were analyzed. The collagen cross-links, pyridinoline (Pyd) and deoxypyridinoline (D-Pyd), were assayed by fluorescence detection after high-performance liquid chromatography (HPLC). Beta2-Microglobulin (beta2M), an index of renal tubular function, was determined by radioimmunoassay. In healthy term infants, urinary collagen cross-links were several times higher than the reported data for older children, with peak values seen at 1 month of age. Excretion of Pyd and D-Pyd was also markedly elevated in 1-month-old preterm infants, despite poor somatic growth. Such high excretion of collagen cross-links probably reflects the state of accelerated bone turnover in infancy. The postnatal change in the cross-links was different from that in urinary beta2M, and the values obtained did not correlate with beta2M in either term or preterm infants. These results indicate that cross-link excretion is not influenced directly by individual renal function.

High-turnover osteopenia in preterm infants: Determination of urinary pyridinium cross-links of collagen

Osteopenia is a frequent condition in preterm infants, but its pathogenesis is uncertain. In the present study, we measured longitudinal changes in the excretion of pyridinium cross-links of collagen (specific markers of bone resorption) and evaluated the relationship between collagen cross-links and other indexes of bone and renal function in preterm infants. In these infants, urinary collagen cross-links were markedly increased on day 7 and day 30 of life and at estimated full-term gestation. The values were several times higher than those of older children and almost comparable to those of healthy full-term infants. Cross-link excretion did not correlate with beta2-microglobulin (B2M) or N-acetyl-beta-D-glucosaminidase (NAG) activity (markers of renal function), indicating that cross-link excretion is not influenced directly by infantile renal function. High serum osteocalcin and low bone mineral density (BMD) in the lumbar spine were also observed at estimated full-term gestation. There was no significant correlation between collagen cross-link excretion and either serum osteocalcin or spine BMD. We conclude that a state of high bone turnover underlies the development of osteopenia in preterm infants.

Selenium status and skeletal tissue metabolism in young infants.

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Urinary excretion of aquaporin-2 in term and preterm infants

Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel of the renal collecting duct and is excreted in human urine. We measured the urinary excretion of AQP-2 by radioimmunoassay in 14 term and 12 preterm infants aged 1 month. Excretion of AQP-2 was low compared with adults, and correlated significantly with urine osmolality in preterm infants. Our results demonstrate that AQP-2 water channels are expressed in the renal collecting duct of both term and preterm infants.

Sugar chain heterogeneity of human urinary chorionic gonadotropin determined by serial lectin affinity chromatography: difference between benign and malignant disease

Human chorionic gonadotropin (hCG) is a glycoprotein of which sugar chains are considered to show structural changes with malignancy. To study the sugar chain heterogeneity of urinary hCG in patients with gynecological disease, we employed serial lectin affinity chromatography (LAC) using concanavalin A (Con A) and phytohemagglutinin-E (PHA-E) which can separate N-glycoside-linked sugar chains, and Jacalin lectin which is specific for O-glycoside-linked sugar chains. The proportion of hCG which did not bind to Con A was clearly higher in patients with cervical cancer than in healthy pregnant women. The complex-type sugar chains bearing bisecting (beta 1-4) N-acetylglucosamine which bound to PHA-E increased in the early stage of cervical cancer, and tri- and tetra-antennary complex type sugar chains also increased in the advanced stages. In addition, the Jacalin-bound hCG increased significantly along with the stage of the cancer, especially in advanced cervical cancer with distant metastases. Taken together, these results show that alteration in sugar chain structures of hCG reflect the advanced stage of cervical cancer.

Prostaglandin A1 enhances c-fos expression and activating protein-1 activity

Prostaglandin A1 (PGA1) increases heat shock element (HSE)-mediated transcription, thereby enhancing expression of HSE-bearing genes, including heat shock proteins. Because we recently found functional HSEs in the human and rodent c-fos promoters, we hypothesized that PGA1 might increase c-fos expression through the HSE. In this study, we revealed that PGA1 induces c-fos expression at least partly by increasing the binding between heat shock factor-1 and the HSE, and that PGA1 enhances activity of activating protein-1 (AP-1). Interestingly, so far as PGA, is present in the medium, AP-1-mediated transcription enhanced by PGA1 cannot be detected by the standard luciferase reporter gene assay. Instead, it can be detected by either checking luciferase mRNA levels in the presence of PGA1 or measuring luciferase activities just after removal of PGA1. These results showed that protein products of some stress-responsive genes can increase, not during the stressful condition, but immediately after recovery from stress.

Evaluation of an immunoassay method for the determination of urinary collagen crosslink excretion.

Pyridinoline (PYD) and deoxypyridinoline (DPYD) are collagen crosslinks found in bone cartilage and extracellular matrix. After th~ collagen is degraded by osteoclasts, the cross links are released into the circulation and eventually excreted into urine. Increase in both PYD and DPYD have been shown to have clinical applicability as specific markers for bone resorption. I A high-performance liquid chromatography (HPLC) method with fluorimetric detection is available for the quantitative measurement of PYD and DPYD in urine. The HPLC method requires extensive sample hydrolyzation and purification steps by cellulose chromatography, making it difficult to measure the utility of PYD and DPYD as biomarkers of bone resorption in routine work. We evaluated a rapid and easy method for measuring free PYD by an enzyme-linked immunosorbent assay (ELISA) method.