Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.

Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.

Active oxygen species generation and cellular damage by additives of parenteral preparations: selenium and sulfhydryl compounds.

We investigated the relationship between active oxygen species (AOS) generation and cultured vascular endothelial cellular damage caused by simultaneous exposure to selenium compounds and sulfhydryl compounds such as cysteine (Cys) or reduced glutathione (GSH). Selenium compounds, selenite, selenate or selenomethionine (SeMet), are added to total parenteral nutrition (TPN) and intravenously administered. We confirmed by luminol dependent chemiluminescence, an indicator of AOS generation, that selenite generates AOS in the presence of clinical concentrations of sulfhydryl compounds, 0.5 mM Cys or 0.5 mM GSH, and that the amount of AOS generated reaches the maximum when their mole ratio is 1:50. However, AOS generation was not observed after simultaneous administration of various concentrations of selenate or SeMet with sulfhydryl compounds. Moreover, simultaneous exposure to 10 microM selenite and sulfhydryl compounds was found to result in significant increases in the [3H]-adenine and lactate dehydrogenase (LDH) release rates from cells, a significant decrease in the amount of cellular protein, and enhancement of cellular damage as compared with after exposure to selenite alone. However, simultaneous exposure to 10 microM selenate or 10 microM SeMet together with sulfhydryl compounds did not induce cellular damage. These findings revealed that selenite generates AOS and causes cellular damage in the presence of sulfhydryl compounds. Accordingly, it seems better to choose selenate or SeMet instead of selenite when a selenium compound is to be added to TPN.

Effects of beta3-integrin blockade (c7E3) on the response to angioplasty and intra-arterial stenting in atherosclerotic nonhuman primates.

Because the beta3-antagonist abciximab (c7E3 Fab) has significantly improved late outcomes after coronary angioplasty, the beta3 integrins have been implicated in the arterial response to injury. However, the mechanisms underlying this benefit are unknown. The observation that c7E3 binds beta3 integrins on vascular cells (alphavbeta3) with affinity equal to that for the platelet glycoprotein IIb/IIIa integrin has led to the hypothesis that c7E3 may act directly on the artery wall to prevent restenosis after angioplasty. To test this hypothesis, we studied the effects of c7E3 on structural changes within the artery wall after angioplasty or stent angioplasty in 23 male cynomolgus monkeys with established atherosclerosis. Animals were randomly assigned to receive either a bolus of c7E3 (0.4 mg/kg IV, n=11) followed by a 48-hour infusion (0. 2 microg. kg-1. min-1) or an equal volume of vehicle (n=12). Animals received weight-adjusted aspirin and heparin and then underwent unilateral iliac artery experimental angioplasty and subclavian artery stent angioplasty (Palmaz). Iliac artery lumen diameter (LD) was determined by angiography at baseline (LDPre), after angioplasty (LDPost), and 35 days later (LDDay35). Arteries were then fixed by perfusion and removed for analysis. Lumen, intima, media, and external elastic lamina (EEL) areas were measured in iliac artery cross sections. Values from each injured iliac artery were normalized to the contralateral uninjured iliac artery to control for interanimal variability in baseline artery size and atherosclerosis extent. Intimal area was also measured in subclavian stent cross sections. c7E3 blocked platelet aggregation and prolonged the bleeding time from 2.8+/-1.1 to 19.8+/-2.5 minutes, P<0.001. Experimental angioplasty increased LDPost an average of 28%, and the initial gain was similar in both groups (P=NS). Despite an anti-platelet effect, c7E3 did not inhibit iliac lumen narrowing (LDDay35-LDPost: c7E3, -0.69+/-0.17 versus vehicle, -0.99+/-.17 mm, P=0.35); intimal hyperplasia (neointima area: c7E3, 1.12+/-.28 versus vehicle, 1.22+/-.20 mm2, P=0.77); or decrease in artery wall size (EEL area [percent of uninjured control]: c7E3, 101+/-7% versus vehicle, 121+/-7%). Stent intimal hyperplasia was also unaltered by c7E3 treatment (neointimal area: c7E3, 1.09+/-0.16 versus vehicle, 1. 28+/-0.11 mm2, P=0.36). These results suggest that the benefits of c7E3 treatment in coronary angioplasty were not from inhibition of intimal hyperplasia or improved artery wall remodeling. Alternative mechanisms should be explored to explain improved late outcomes after angioplasty in patients treated with c7E3.

Cross-reactivity to olive tree pollen and orchard grass pollen in patients with pollinosis.

We studied 92 patients with allergic rhinitis in Syodoshima, Japan, during the pollen season between April and June to evaluate the cross-reactivity to different antigens, including pollen from the olive tree (Olea europaea) and orchard grass (Dactylis glomerata). Olive tree pollen was present in the atmosphere for 23 days, from May 19 to June 12, 1994. Specific IgE antibodies for olive tree pollen antigen were present in 21 (26.9%) of the 78 patients with allergic rhinitis. Nine (24.3%) of the 37 patients with allergic rhinitis exhibited positive skin reactivity to an extract of olive tree pollen. Fifteen (88.2%) of the 17 patients who had IgE reactivity in their sera to olive tree pollen antigen demonstrated allergic reactions to an extract of olive tree pollen. Specific IgE antibodies for orchard grass pollen antigen were present in 43 (48.3%) of the 89 patients with allergic rhinitis and 20 (95.2%) of the 21 patients who had IgE reactivity in their sera to olive tree pollen antigen. The inhibition test using the CAP System revealed that the reactivity of the IgE antibody specific for olive tree pollen antigen was inhibited dose-dependently by an extract of orchard grass pollen. These findings show that there is a reaction in some patients with grass (Gramineae) pollinosis that might be induced by olive tree pollen.

Selenium administration to a ten-year-old patient receiving long-term total parenteral nutrition (TPN)--changes in selenium concentration in the blood and hair.

Muscle pain in the lower limbs occurred in a child with short bowel syndrome who has been receiving longterm total parenteral nutrition (TPN). Biochemical parameters revealed that the plasma and erythrocyte selenium concentrations were below the normal range for children and intravenous injection of selenium prepared from selenious acid was started at a dose of 100 micrograms per day. Muscle pain in the lower limbs disappeared one month afterwards. At this point in time, the elevation of the plasma selenium concentration was noted but the erythrocyte selenium concentration remained low. When administration was suspended due to catheter-induced fever five months later, the whole blood selenium concentration decreased again and the symptoms recurred. Accordingly, the dose of selenium was increased to 200 micrograms/day. Subsequently, the blood selenium concentration recovered to the normal range for children. After the dose increase to 200 micrograms/day, concentrations in hair samples collected at every centimeter distance from the root end were determined. The selenium concentration at the root end was found to be higher than the normal range for children, indicating that this was an excessive dose case. Although the dose was decreased from 200 micrograms/day to 120 micrograms/day, the plasma and erythrocyte selenium levels did not go down. Furthermore, the selenium level in the hair reached a plateau, and no recurrence of symptoms was observed. The above results indicate the usefulness of monitoring the selenium concentration in hair in addition to determining the blood selenium level and GSH-Px activity in administering selenium to children undergoing TPN.

Intestinal absorption of oyster shell electrolysate.

High voltage electric current was passed through oyster shell powder for electrolysis. The crystalline shape of oyster shell electrolysate appeared to be quite different from that of CaO or CaCO3. Higher serum calcium values were achieved by oral administration of the same amount of as oyster shell electrolysate than as calcium carbonate in vitamin D-deficient rats, suggesting a better intestinal absorption of the former than the latter. In four patients with postoperative hypoparathyroidism with reduced intestinal calcium absorption, the same amount of elementary calcium as oyster shell electrolysate was more effective than calcium carbonate in raising serum calcium in the absence of vitamin D supplement. Oyster shell electrolysate was also more effective in suppressing serum parathyroid hormone concentration than calcium carbonate in two patients with secondary hyperparathyroidism with renal failure. Calcium thus appears to be more readily absorbed from oyster shell electrolysate than from calcium carbonate through intestinal barriers produced by insufficient vitamin D action.

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium.

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.

Biological activities and binding properties of 23,23-difluoro-25-hydroxyvitamin D3 and its 1 alpha-hydroxy derivative.

23,23-Difluoro-25-hydroxyvitamin D3 is 5-10 times less active than 25-hydroxyvitamin D3 in stimulating intestinal calcium transport, bone calcium mobilization, increasing serum phosphorus, mineralization of rachitic bone, and binding to the plasma transport protein in rats. It is converted to 23,23-difluoro-1 alpha, 25-dihydroxyvitamin D3 by chick renal 25-hydroxyvitamin D-1-hydroxylase. This compound is one-seventh as active as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Thus, fluoro substitution on carbon-23 of vitamin D has an unexpected and unexplained suppressive action on plasma binding and biological activity. However, since this substitution does not block the biological response of 25-hydroxyvitamin D3, these results provide additional evidence that 23-hydroxylation of vitamin D is not involved in biological function.