Evidence of Mucin Secretion in Human Lung Adenocarcinoma Cell Lines NCIH650 and NCIH2077 and Effect of Select Secretagogues on Mucin Secretion

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor ³H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.     

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome

Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.     

Antizyme is a target of sex-lethal in the Drosophila germline and appears to act downstream of hedgehog to regulate sex-lethal and cyclin B

The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early germ cells in Drosophila melanogaster. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of Ornithine Decarboxylase Antizyme, was isolated. gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. We also show that Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis.     

Can Dreams During Pregnancy Predict Postpartum Depression?

Postpartum depression (henceforth PPD) is an emotional disturbance which occurs in as much as 20% of the childbearing population. This study attempted to ascertain whether dreams, offering unconscious expression of internal emotional processes, could help to identify early signs of PPD. It was hypothesized that differences would be found in the emotional dream work of pregnant women who either later developed or did not develop PPD. 166 women participated in the two stages of the study. During stage I, the women were interviewed in the last trimester of their first pregnancies. The interview included a demographic questionnaire and an account of a dream. During stage II, the women were interviewed 6–10 weeks after giving birth. The second interview included only the EPDS scale for affirming or denying the occurrence of PPD. The findings of the study confirm the hypothesis that dreams of pregnant women can differentiate among women who are or are not at-risk for PPD. It was found that more unpleasant dreams and dreams expressing apprehension were found among women who did not later develop PPD, than among women who did develop PPD.     

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.     

The regulation of death-associated protein (DAP) kinase in apoptosis

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.     

Amphiphilic block copolymers as bile acid sorbents: 1. Synthesis of polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride)

The systematic investigation of the synthesis of polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) was accomplished by employing both polystyrene-b-poly(tert-butyl acrylate) and its hydrolyzed derivative, polystyrene-b-poly(acrylic acid) (PS-b-PAA) as starting materials, and coupling them with N,N-dimethylethylenediamine (DMED). The various reactions and intermediates we examined include aluminum amides, acid chlorides, and imides derived from carbodiimides, all in a variety of solvents. We present below our investigation of several synthetic routes and conclude that the carbodiimide coupling of PS-b-PAA with DMED followed by quaternization and counterion exchange is the most effective method of achieving the target. A brief discussion of the merits of each procedure in the context of block copolymers is given, and IR spectroscopic evidence for the postpolymerization synthesis of the poly(acrylamide) block is provided.     

Simultaneous effect of calcium,magnesium, copper and cobalt ions on sapogenin steroids content in callus cultures of Agave amaniensis

The simultaneous effect of calcium, cobalt, copper and magnesium ions and their interactions on growth and sapogenin steroids accumulation in callus cultures of Agave amaniensis was studied by using a central composite second-order rotatable design. The absence of calcium ions in media increased the sapogenin steroid content, while relatively high concentration of magnesium, cobalt and copper ions simultaneously inhibited the sapogenin steroid formation. This revised version was published online in June 2006 with corrections to the Cover Date.