Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: synthesis and biological activity

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene (O-C(2)), O-butylene (O-C(4)), and methylene (C(1)) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting human lymphoblastic leukemic cells (CCRF-CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis (2-chloroethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lymphoblastic leukemic cells (CCRF-CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or Taxol-resistant (CCRF-CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the Taxol therapeutic dose [i.e., 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5); intravenous injection] on nude mice bearing human breast carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft tumor suppression of 81-96% using human T-cell acute lymphoblastic leukemia CCRF-CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma SK-OV-3 tumor models.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

The analysis of reaction norms for age and size at maturity using maturation rate models

Reaction norms for age and size at maturity are being analyzed to answer important questions about the evolution of life histories. A new statistical method is developed in the framework of time-to-event data analysis, which circumvents shortcomings in currently available approaches. The method emphasizes the estimation of age- and size-dependent maturation rates. Individual probabilities of maturation during any given time interval follow by integrating maturation rate along the growth curve. The integration may be performed in different ways, over ages or sizes or both, corresponding to different assumptions on how individuals store the operational history of the maturation process. Data analysis amounts to fitting generalized nonlinear regression models to a maturation status variable. This technique has three main advantages over existing methods: (1) treating maturation as a stochastic process enables one to specify a rate of maturation; (2) age and size at which maturation occurs do not have to be observed exactly, and bias arising from approximations and interpolations is avoided; (3) ages at which sizes are measured and maturation status are observed can differ between individuals. An application to data on the springtail Folsomia candida is presented. Models with age-dependent integration of maturation rates were preferred. The analysis demonstrates a significant size dependence of the maturation rate but no age dependence.     

Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse

The importance of understanding the dynamics of DNA condensation is inherent in the biological significance of DNA packaging in cell nuclei, as well as for gene therapy applications. Specifically, the role of ligand hydrophobicity in DNA condensation has received little attention. Considering that only multivalent cations can induce true DNA condensation, previous studies exploring monovalent lipids have been unable to address this question. In this study we have elucidated the contribution of the hydrophobic effect to multivalent cation- and cationic lipid-DNA binding and DNA collapse by studying the thermodynamics of cobalt hexammine-, spermine-, and lipospermine-plasmid DNA binding at different temperatures. Comparable molar heat capacity changes (DeltaC(p)) associated with cobalt hexammine- and spermine-DNA binding (-23.39 cal/mol K and -17.98 cal/mol K, respectively) suggest that upon binding to DNA, there are insignificant changes in the hydration state of the methylene groups in spermine. In contrast, the acyl chain contribution to the DeltaC(p) of lipospermine-DNA binding (DeltaC(p ) = DeltaC(p lipospermine) - DeltaC(p spermine)) is significant (-220.94 cal/mol K). Although lipopermine induces DNA ordering into "tubular" suprastructures, such structures do not assume toroidal dimensions as observed for spermine-DNA complexes. We postulate that a steric barrier posed by the acyl chains in lipospermine precludes packaging of DNA into dimensions comparable to those found in nature.     

The ontogeny of the urogenital system of the spotted hyena (Crocuta crocuta Erxleben)

Studies were conducted to elucidate the importance of androgen-mediated induction of the extreme masculinization of the external genitalia in female spotted hyenas. Phallic size and shape; androgen receptor (AR) and alpha-actin expression; and sex-specific differences in phallic retractor musculature, erectile tissue, tunica albuginea, and urethra/urogenital sinus were examined in male and female fetuses from Day 30 of gestation to term. Similar outcomes were assessed in fetuses from dams treated with an AR blocker and a 5alpha-reductase inhibitor (antiandrogen treatment). Clitoral and penile development were already advanced at Day 30 of gestation and grossly indistinguishable between male and female fetuses throughout pregnancy. Sex-specific differences in internal phallic organization were evident at Gestational Day 45, coincident with AR expression and testicular differentiation. Antiandrogen treatment inhibited prostatic development in males and effectively feminized internal penile anatomy. We conclude that gross masculinization of phallic size and shape of male and female fetuses is androgen-independent, but that sexual dimorphism of internal phallic structure is dependent on fetal testicular androgens acting via AR in the relevant cells/tissues. Androgens secreted by the maternal ovaries and metabolized by the placenta do not appear to be involved in gross masculinization or in most of the sex differences in internal phallic structure.     

The high-resolution architecture and structural dynamics of Bacillus spores

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.     

Mechanical properties of Xenopus egg cytoplasmic extracts

Cytoplasmic extracts prepared from Xenopus laevis eggs are used for the reconstitution of a wide range of processes in cell biology, and offer a unique environment in which to investigate the role of cytoplasmic mechanics without the complication of preorganized cellular structures. As a step toward understanding the mechanical properties of this system, we have characterized the rheology of crude interphase extracts. At macroscopic length scales, the extract forms a soft viscoelastic solid. Using a conventional mechanical rheometer, we measure the elastic modulus to be in the range of 2-10 Pa, and loss modulus in the range of 0.5-5 Pa. Using pharmacological and immunological disruption methods, we establish that actin filaments and microtubules cooperate to give mechanical strength, whereas the intermediate filament cytokeratin does not contribute to viscoelasticity. At microscopic length scales smaller than the average network mesh size, the response is predominantly viscous. We use multiple particle tracking methods to measure the thermal fluctuations of 1 microm embedded tracer particles, and measure the viscosity to be approximately 20 mPa-s. We explore the impact of rheology on actin-dependent cytoplasmic contraction, and find that although microtubules modulate contractile forces in vitro, their interactions are not purely mechanical.     

Molecular and quantitative genetic divergence among populations of house mice with known evolutionary histories

Evolutionary biologists have long been interested in the processes influencing population differentiation, but separating the effects of neutral and adaptive evolution has been an obstacle for studies of population subdivision. A recently developed method allows tests of whether disruptive (ie, spatially variable) or stabilizing (ie, spatially uniform) selection is influencing phenotypic differentiation among subpopulations. This method, referred to as the F(ST) vs Q(ST) comparison, separates the total additive genetic variance into within- and among-population components and evaluates this level of differentiation against a neutral hypothesis. Thus, levels of neutral molecular (F(ST)) and quantitative genetic (Q(ST)) divergence are compared to evaluate the effects of selection and genetic drift on phenotypic differentiation. Although the utility of such comparisons appears great, its accuracy has not yet been evaluated in populations with known evolutionary histories. In this study, F(ST) vs Q(ST) comparisons were evaluated using laboratory populations of house mice with known evolutionary histories. In this model system, the F(ST) vs Q(ST) comparisons between the selection groups should reveal quantitative trait differentiation consistent with disruptive selection, while the F(ST) vs Q(ST) comparisons among lines within the selection groups should suggest quantitative trait differentiation in agreement with drift. We find that F(ST) vs Q(ST) comparisons generally produce the correct evolutionary inference at each level in the population hierarchy. Additionally, we demonstrate that when strong selection is applied between populations Q(ST) increases relative to Q(ST) among populations diverging by drift. Finally, we show that the statistical properties of Q(ST), a variance component ratio, need further investigation.