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ABSTRACT. The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4°C. Following incubation of these modified cells at 37°C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37°C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37°C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.

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Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.

N-(p-Coumaroyl)serotonin (C) and N-feruroylserotonin (F) with antioxidative activity are present in safflower oil. The protective effects of C and F were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored by an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The rate of recovery of LVDP from ischemia by reperfusion was 30.8% in the control, while in the presence of C or F a gradual increase to 63.2 or 61.0% was observed. Changes of transient NO signals (TNO) released from heart tissue in one contraction (LVDP) were observed to be upside-down with respect to transient fura-2-Ca2+ signals (TCa) and transient O2 signals detected with a pO2 electrode. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and the release of NO increased with no twitching and remained at a high steady level. The addition of C increased the NO level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. On reperfusion, the increased diastolic level of TCa and TNO returned rapidly to the control level with the recovery of LVDP. By in vitro EPR, C and F were found to directly quench the activity of active radicals. Therefore, it is concluded that the antioxidant effects of two derivatives isolated from safflower play an important role in ischemia-reperfusion hearts in close relation with NO.

Key words: SV40/cell membrane markers/endocytotic vacuoles/nuclear envelope/diaphragm/interaction ABSTRACT. The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4°C, and then chased for 2 h at 37°C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membraneof some vacuoles seemedto be in contact with the outer nuclear membrane.Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membraneswere often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4°C, chased for 2 h at 37°C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membraneas well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane,and further indicate that someendocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.

In vitro bone formation by cells derived from adult rabbit femurs was investigated on or in several substrates with small porous hydroxyapatite granules (HAGs). When the bone fragments were cultured in HAG-packed glass tubes, which were inclined (5 degrees -30 degrees ) and rotated 90 degrees per day after one week of culture, thin lamellar tissues were newly formed in narrow spaces among the HAGs. By 11 days of culture, these tissues had been mineralized except for their periphery and had well developed collagen bundles and several monolayer cells. Some cells resided in bone lacuna-like spaces. By contrast, mineralization was negligible in 6-week cultures on two-dimensional glass and polystyrene plates with or without two-dimensionally arranged HAGs on their surfaces and in three-dimensional collagen gels with or without HAGs in spite of active cell proliferation. These results suggest that osteogenesis is accelerated in a specific three-dimensional constitution of extracellular matrix and/or under the effects of mechanical forces for the new tissue and that bioactive HAGs offer favorable three-dimensional spaces for osteogenic tissue formation.