TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Colposacropexy: an alternative technique

Colposacropexy procedures restore anatomically correct apical vaginal support on the levator plate at the ischial spine level. Venous hemorrhage resulting from laceration of presacral veins during suture fixation is the major hazard of this procedure. Titanium orthopedic bone anchor fixation minimizes this risk through precision placement of the bone anchor-suture unit.     

Implant-supported prostheses for treatment of adults with cleft palate

Six adult patients with cleft palate, ranging in age from 47 to 78 years, were treated with self-tapping titanium implants. Twenty-three implants, 7 to 15 mm in length, were placed. Of these, one (4%) was 7 mm, eight (35%) were 10 mm, nine (39%) were 13 mm, and five (22%) were 15 mm. Time between stage I and stage II implant surgeries was 5 to 14 months, averaging 8.3 months. Time from stage II surgery to the present is 1.5 to 5 years, averaging 3 years. Of the 23 implants placed, 21 (91%) achieved osseointegration. One (4%) implant was not used prosthetically. Two (9%) 10 mm implants failed to integrate in one patient. All patients were treated with a maxillary complete denture or overdenture. Five (83%) required the addition of a pharyngeal section for speech enhancement.     

Correlation between leukotriene D4-induced contraction and cytosolic calcium elevation: a quantitative and simultaneous evaluation in smooth muscle

The role of Ca++ as an intracellular messenger in leukotriene (LT)D4-induced muscle contraction was investigated by measuring force development and elevation in cytosolic Ca++ concentration simultaneously in strips of guinea pig ileal longitudinal muscle loaded with the fluorescent calcium indicator Fura 2. Upon addition of LTD4, a simultaneous increase in tension and cytosolic calcium concentration, [Ca++]i, was observed. Cumulative applications of LTD4 induced concentration-dependent increases in both muscle tension and [Ca++]i, being the half-maximal effect reached at approximately 6 to 9 nM. A statistically significant positive correlation (r = 0.993, P < .001) exists between the two parameters examined. Removal of calcium in the bathing solution, accompanied by addition of 7.5 mM EGTA, completely prevented any increase in either calcium levels or force development, thus indicating a role for Ca++ influx, rather than a release from intracellular stores. All of the LTD4 antagonists tested were able to counteract the effect of the leukotriene on both [Ca++]i and tension increase. However, although LY171883 shifted both of the LTD4 curves to the right in a parallel fashion, FPL 55712 and ICI 198,615 behaved as non-competitive antagonists in reversing the effect of LTD4 on [Ca++]i and tension. Thus, these results strongly suggest that changes in muscle tension induced by LTD4 are attributable to changes in cytosolic free Ca++ concentrations in guinea pig ileum.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.