Phosphorylation of a constrained azacyclic FTY720 analog enhances anti-leukemic activity without inducing S1P receptor activation.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts.

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.

Regulation of beta-catenin levels and localization by overexpression of plakoglobin and inhibition of the ubiquitin-proteasome system.

beta-Catenin and plakoglobin (gamma-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and alpha-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, beta-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increased the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolished beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.

Role of stress fibers in the association of intermediate filaments with microtubules in fibroblast cells.

The association between intermediate filaments (IF) and microtubules (MT) has been demonstrated by several experiments using MT inhibitors and by microinjecting specific antibodies. The actin cytoskeleton has recently been assigned a role in this process of drug induced IF collapse. However, this was not found to be true in large cells with irregular morphology. For instance, in early passage diploid fibroblasts of human origin and in armadillo cell lines, where the cells are large, irregular in shape and exhibit prominent stress fibers (SF), depolymerization of MT with nocodazole did not lead to collapse of IF. Instead, the IF formed bundles of coils that seemed to associate with the SF. Disintegration of the SF with cytochalasin B led to the collapse of the IF. It appears that the actin organization in such large cells with extensive SF, is not as contractile as in typical spindle shaped fibroblasts which have relatively less stable actin organization. The stable SF may actually prevent IF collapse.

Regulation of cytoplasmic microtubule complex organisation in somatic cell hybrids.

Regulation of cytoplasmic microtubule complex (CMTC) organization was studied in cultured human fibroblasts and mouse macrophages by somatic cell fusion. The heterokaryons stained with antitubulin antibody had fibroblast-like CMTC even 72 hours after fusion. There was no change in CMTC pattern when more than one macrophage had fused with one fibroblast. However, the macrophage CMTC was expressed in heterokaryons when the former were located at the periphery of the heterokaryon. To evaluate the role of existing CMTC in determining the CMTC of heterokaryons, the heterokaryons were treated with nocodazole to depolymerize the CMTC and then allowed to recover. The resultant CMTC was fibroblast like.

Reconstruction of Indian life tables for 1901-1981 and projections for 1981-2001.

"An attempt has been made to reconstruct life tables for India from 1901-11 to 1971-81 and to project for the decades 1981-91 and 1991-2001 by adopting a Brass relational model. The earlier actuarial life tables seem to have been based on a British model of sex differentials in mortality leading to higher life expectancies for females--not in tune with Indian experience. Consistency has been attained in this study by taking recourse to mortality patterns obtained from the Sample Registration System."

Estimating child mortality and modelling its age pattern for India.

"Using data [for India] on proportions of children dead...estimates of infant and child mortality are...obtained by Sullivan and Trussell modifications of [the] Brass basic method. The estimate of child survivorship function derived after logit smoothing appears to be more reliable than that obtained by the Census Actuary. The age pattern of childhood mortality is suitably modelled by [a] Weibull function defining the probability of surviving from birth to a specified age and involving two parameters of level and shape. A recently developed linearization procedure based on [a] graphical approach is adopted for estimating the parameters of the function."

Developing model schedules of female proportions single for India.

The Indian Census provides marital status data cross classified by sex and age. There are, however, several limitations in the general state of information and the quality of data on marital status. For instance, the ratios proportions single in corresponding age groups in 2 consecutive censuses have been found to exceed unity in the absence of any significant migration. In the present study, the authors adjust the the schedules of proportions single for 1901-1971 with a logit linear model. In spite of the serious nature of errors in the data, the model appears to work well. In assessing its quality, the technique is applied to more accurate Japanese data, and found to have an extremely good fit. A comparison with the Coale-McNeil nuptiality model reveals that for the Indian situation the logit model may be more appropriate.

Demography of sterilization: Indian experience.

Using program statistics and a few local studies, the paper examines the prevalence of sterilization in India and makes demographic analysis of the performance. Major points of note are that the prevalence rates expressed in terms of currently married women showed irregular fluctuations across time and the age pattern of acceptance hardly agreed with that of the married population. The selectivity of high fertility couples for sterilization had been fairly high, resulting in a low fertility impact. Estimates of births averted due to the program were derived by using a computerized microsimulation model CONVERSE. It appears that the path of fertility changes was not smooth in the last decade and the impact has been, as a rule, greatest 2 years after acceptance.

Path analysis of areal fertility in rural India.

PIP: This study illustrates the application of a recursive path analytic model in analyzing how social and economic factors condition the relationship between fertility and acceptance of birth control measures. It uses States and Union Territories of India (rural) as units of observation. The data used for each area is current fertility measures of non-program variables having a direct or indirect effect on fertility. The whole set of concomitant variables could not be considered, as they are not all to be found in the usual sources of areal statistics. Corresponding to the model, tentatively proposed, the path diagram has been used to represent dependence of endogenous variabels upon other endogenous variables, exogenous variables, or disturbances. A few tests were done to ensure that the model was appropriate. For example, the assumption of no direct effect of a variable on certain others was checked by comparing correlations obtained from the model with those observed. Several results emerge from the analysis. For example, age at marriage influences age specific fertility through its effect on marital fertility and to a lesser extent through proportion married. Acceptance of birth control measures decreases age specific fertility primarily through marital fertility. The study breaks down the composite variable (age specific fertility) into its components (proportion married and marital fertility) before conducting a search for its variability. The results indicate that the national antinatalist policy is unlikely to contribute much to acceleration of fertility decline. With socioeconomic development and institution of social measures such as raising the minimum age at marriage, motivations to accept effective control measures at a low parity level will take place and fertility will automatically move downward.^ieng

Data base of abortion investigation: methodological issues.

PIP: A critical examination of data base of abortion information is made. Estimates of incidence rate that may emerge from these sources are indicated. From the knowledge of the distribution of "life" of pregnancy over time, a procedure to estimate the prevalence of foetal wastage is given. Based on the criterion of minimum variance, the abortion indicator measured in terms of pregnancies appears to be technically better than other ratios. Relation between abortion rate and birth rate is discussed. It is shown that in a limited period if the rate of increase in birth rate is made equal to the fall of the abortion ratio, the whole of the change in birth rate will be passed on to the abortion rate.^ieng

Population estimates for Bangladesh: The use of a specific transitional population model.

Summary The development of population in Bangladesh was affected by a succession of man-made and natural calamities, such as the Bengal Famine of 1943, refugee movements following the partition of India and Pakistan in 1947, devastating floods and cyclones around 1970, and the military action during the war of liberation. Though there had been a tradition of census taking and vital registration in Bangladesh, as part of the Indian sub-continent, extending for over a century, vital registration was so deficient as to be almost valueless, and there were gross misstatements of age and under-enumeration in the censuses. In the census of 1941, on the other hand, political manoeuvring led to a substantial overcount of the population. In this paper, Bangladesh population trends are studied within the broader framework of the subcontinent, taking account of plausible differentials. A considerable element of uncertainty was introduced into growth trends as a result of variations in the completeness of census-taking and of unrecorded refugee and labour movements across open land borders. In this connection the substantial inflationary bias associated with techniques of population estimation using the dual record system is discussed. The application of stable population models is even less justified in Bangladesh with its history of declining mortality. A transitional age structure model was constructed on the basis of the information available on declining mortality and accelerating growth and the model was made even more specific by modifications which took care of the impact of recent calamities and of unrecorded migration. The population base of the census of 1961 was adjusted in accordance with this model. The local mortality age pattern was used in projecting the population by sex and age groups to the date at which the census was originally due to be taken in 1971, and to the date when it was actually taken in March 1974. The post-1970 calamities and their effect on mortality were ignored. The aggregate estimate of population of 72.9 million in March 1974 is slightly in excess of the census count (by about two per cent) reported provisionally as 71.3 million. The excess in our estimate could be accounted for by the losses due to cyclone and military action.