Cellular depletion of p56lck during thymocyte apoptosis

The src-related protein tyrosine kinase p56lck is thought to be important in regulating maturation and functional responsiveness of T cells and thymocytes. In the present studies we report that expression of p56lck is suppressed during apoptosis. Using primary cultures of rat thymocytes, we found that agents that are effective in inducing apoptosis, including okadaic acid, dexamethasone, and antibodies to the CD3 receptor, also deplete cells of p56lck. This process is rapid, occurring within 24 h, and is not due to cytotoxicity. Inhibition of DNA fragmentation in apoptotic cells with the endonuclease inhibitor ZnCl2 failed to prevent depletion of p56lck, suggesting that it was not a consequence of the DNA degradation process. Using the thymic lymphoma cell line LSTRA, apoptosis was also associated with cellular depletion of p56lck. In contrast to thymocytes, this process required 48-72 h possibly because these cells overexpress p56lck. Although at this time we are uncertain as to the precise role of p56lck in the process of apoptosis, our results indicate that changes in the expression of this protein in thymocytes is an important marker of programmed cell death.     

Conformal proton therapy for prostate carcinoma

The suppression of apoptosis may contribute to the carcinogenicity of the peroxisome proliferators (PPs), a class of non-genotoxic rodent hepatocarcinogens. Our previous work demonstrated that the PP nafenopin suppressed both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced hepatocyte apoptosis both in vivo and in vitro. Here, we extend these observations by demonstrating the ability of nafenopin to suppress apoptosis induced by other major candidates for the signalling of cell death in the liver. Treatment of rat or mouse hepatocyte monolayers with TGFbeta1 or the DNA damaging drugs etoposide or hydroxyurea induced high levels of apoptosis. Western blot analysis did not support a role for either p53 or p21waf1 in etoposide-induced apoptosis in rat hepatocytes. Treatment of mouse hepatocytes with an agonistic anti-Fas antibody also resulted in an induction of high levels of apoptosis. Pre-addition and continued exposure to nafenopin suppressed apoptosis induced by all three stimuli. Overall, our studies demonstrate that the ability of nafenopin to protect hepatocytes from apoptosis is not restricted to species or apoptotic stimulus. It is possible, therefore, that the PPs may suppress apoptosis by acting on diverse signalling pathways. However, it seems more likely that nafenopin suppresses hepatocyte apoptosis elicited by each death stimulus by impinging on a core apoptotic mechanism.     

Early child-care selection: variation by geographic location, maternal characteristics, and family structure

More than half of all U.S. infants and toddlers spend at least 20 hr per week in the care of a nonparent adult. This article uses survival analysis to identify which families are most likely to place their child in care and the ages when these choices are made, using data from a national probability sample of 2,614 households. Median age at first placement is 33 months, but age varies by geographic region, mother's employment status during pregnancy, mother's education level, and family structure (1 vs. 2 parents, mother's age at 1st birth, and number of siblings). Controlling for these effects, differences by race and ethnicity are small. Implications for studies of child-care selection and evaluations of early childhood programs are discussed.     

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.     

Life table study of Aedes aegypti (Diptera: Culicidae) in Puerto Rico fed only human blood versus blood plus sugar

Life table studies were performed in 1996 with Aedes aegypti (L.) during the low (cool/dry) and high (hot/rainy) dengue virus transmission seasons in Puerto Rico. Mated adult females from field-collected pupae were placed individually in cages and divided into 2 treatment groups: one was fed only human blood and the other human blood plus a 10% sucrose solution. Survival and number of eggs laid were recorded daily for each female. During both seasons, age specific survivorship was higher for the blood plus sugar group, groups fed only human blood had higher reproductive outputs (mx), and net replacement rates (Ro) for blood only groups were higher than for those fed blood plus sugar. Intrinsic rates of growth (r) were the same for both treatments during the low (cool/dry) transmission season, but higher for the blood-only treatment during the high (hot/rainy) transmission season. Our results indicate that feeding on only human blood provides an evolutionary advantage to Ae. aegypti females in Puerto Rico. These results are similar to those from an earlier study carried out with Ae. aegypti in Thailand; the advantage of feeding on human blood does not seem to be restricted to a particular geographic region. We also found that the benefits associated with human feeding persist through epidemiologically different times of the year. We conclude that feeding on human blood is reproductively beneficial for Ae. aegypti, which may increase their contact with human hosts, and therefore may influence their vectorial capacity for dengue viruses through frequent feeding on blood.     

Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis

BACKGROUND AND OBJECTIVE: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a new method which can be used to study cryoprecipitates from the sera of cryoglobulinemic patients. It led to the identification of a new type of cryoprecipitate, tentatively named II-III, characterized by polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM. Some discrepancies with the conventional classification of cryoglobulins were revealed. The association of particular clinical features with the classification of cryoglobulins by 2-D PAGE is examined. DESIGN AND METHODS: Sixty consecutive patients affected by cryoglobulinemic syndrome with mixed cryoglobulins were included in the study. All patients were evaluated for cutaneous, articular, hepatic, renal and nervous involvement. The washed cryoprecipitates were typed using both techniques: immunofixation electrophoresis (IFE) and 2-D PAGE. RESULTS: Sixteen (6 cases of type II and 10 of type III by IFE) of 60 cryoprecipitates (26.6%) appeared as type II-III by 2-D PAGE analysis. Nine cases were classified differently by IFE and 2-D PAGE. Mixed cryoglobulins of type II-III were not associated with a particular clinical pattern. Examining the clinical findings in the mono group (those with monoclonal IgM alone) and the poly group (those with polyclonal IgM alone or polyclonal and monoclonal IgM) we found clearly significant differences: more severe liver involvement in the poly group, and higher cryocrit and creatinine values, lower C4 level and more severe purpura in the mono group. INTERPRETATION AND CONCLUSIONS: Our results confirm the reliability of 2-D PAGE in characterizing cryoprecipitates. This sensitive method can demonstrate a higher number of monoclonal components, undetectable by IFE. Type II-III cryoglobulins are not associated with a particular clinical pattern. The presence or absence of polyclonal IgM in mixed cryoglobulins seems to be correlated with some clinical findings.     

Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound

We have synthesized a new compound, 3-bromoacetylamino benzoylurea (3-BAABU), which showed strong cancericidal activity by inducing irreversible mitotic arrest and subsequently apoptosis in human T cell leukemic cells (CEM), human biphenotypic leukemic cells (SP), a human prostate cancer cell line (PC-3), murine melanoma cells (B-16), and murine lymphoma/leukemia cells (EL4) in vitro with an ID50 in the range of 0.013-0.07 microg/ml (0.04-0.22 microM). Treatment of tumor cells for 12-24 h with 3-BAABU resulted in mitotic arrest at prometaphase/metaphase/anaphase, with separation and dispersion of chromosomes and with the absence of mitotic spindle apparatus in cytoplasm. Treatment with 3-BAABU had no cytotoxic and mitotic blocking effect in normal human lymphocytes, proliferating fibroblast cells (3T3), or proliferating myocardial cells (MOT). Cell cycle analyses showed that most treated leukemic cells accumulated at M phase 12 h after treatment. By the end of 48 h of treatment, the cells underwent apoptosis with DNA fragmentation. 3-BAABU inhibited the assembly of microtubules from tubulin but did not interfere with the disassembly of microtubules. The presence and the position of bromine and urea groups on the benzoic ring are the determining factors for its inhibition of microtubule assembly. Replacing bromine with chlorine yielded much less mitotic blocking activity and increased the ID50 40-fold. Substitution of the urea group with ethyl ester abrogated the activity of blocking mitosis but induced apoptosis. Moving the bromoacetylamino group from the 3-position to the 4-position removed blocking activity for mitosis but induced necrosis. These results suggest that 3-BAABU possesses a unique and functional structure and is a potential agent for cancer chemotherapy.