Paradoxes in exogenous androgen treatments of bluegill

Three experiments were conducted to investigate the effect of oral administration of trenbolone acetate and 17a-methyltestosterone on sex determination in 28-day-old bluegill, Lepomis macrochirus Rafinesque, fry. Multiple androgen doses and various treatment durations were tested. All treatments produced fewer males and females than did the control group (P < 0.01). All concentrations of both androgens produced a high proportion of intersex fish (38–81%). The number of males and females declined with the increase in androgen dose or treatment duration. Sterile fish were found in treatments with a higher dose rate or a longer treatment period. The predominance of intersex fish and reductions of both males and females in the androgen treatments suggest that gonadal development of both genotypic male and female fish were being altered by the hormone treatment.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Age-specific differences in oncogenic pathway deregulation seen in human breast tumors

Purpose

To define the biology driving the aggressive nature of breast cancer arising in young women.

Experimental Design

Among 784 patients with early stage breast cancer, using prospectively-defined, age-specific cohorts (young ≤45 years; older ≥65 years), 411 eligible patients (n = 200≤45 years; n = 211≥65 years) with clinically-annotated Affymetrix microarray data were identified. GSEA, signatures of oncogenic pathway deregulation and predictors of chemotherapy sensitivity were evaluated within the two age-defined cohorts.

Results

In comparing deregulation of oncogenic pathways between age groups, a higher probability of PI3K (p = 0.006) and Myc (p = 0.03) pathway deregulation was observed in breast tumors arising in younger women. When evaluating unique patterns of pathway deregulation, a low probability of Src and E2F deregulation in tumors of younger women, concurrent with a higher probability of PI3K, Myc, and β-catenin, conferred a worse prognosis (HR = 4.15). In contrast, a higher probability of Src and E2F pathway activation in tumors of older women, with concurrent low probability of PI3K, Myc and β-catenin deregulation, was associated with poorer outcome (HR = 2.7). In multivariate analyses, genomic clusters of pathway deregulation illustrate prognostic value.

Conclusion

Results demonstrate that breast cancer arising in young women represents a distinct biologic entity characterized by unique patterns of deregulated signaling pathways that are prognostic, independent of currently available clinico-pathologic variables. These results should enable refinement of targeted treatment strategies in this clinically challenging situation.     

Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.     

Phylogenetic analysis of cytochromeb sequences in the dasyurid marsupial subfamily phascogalinae: Systematics and the evolution of reproductive strategies

We report DNA sequence variation in 861 bp of the mitochondrial cytochromeb gene from 10 species of the dasyurid marsupial subfamily Phascogalinae (including the New Guinean genusMurexia) and an outgroup planigale (Planigale ingrami). Phylogenetic analyses of these sequences indicate that (1) the subfamily consists of three major clades corresponding to (a)Phascogale, (b) AustralianAntechinus, and (c) New Guinean Antechinus andMurexia; (2) Antechinus habbema constitutes the earliest branch of the New Guinean clade; and (3); Antechinus melanurus and A. naso are sister species within the New Guinean clade. Among Australian antechnuses,A. stuartii andA. swainsonii are more closely related to each other than either is toA. flavipes, a result that is seemingly at odds with all previous systematic studies. Although resolution is limited, it appears thatAntechnius andMurexia species form a clade to the exclusion ofPhascogale. This relationship suggests that male semelparity is not a strong synapomorphy for Australian antechinuses and phascogales, despite its apparent physiological similarity in the two groups.To whom correspondence should be addressed.     

Metal concentrations in surficial sediments from hypersaline lakes,Australia

A major drawback to the use in aquaculture of members of the Eubranchiopoda from temporary pool environments is that their eggs do not hatch readily. An investigation of the factors influencing the hatching of eggs of the fairy shrimpStreptocephalus macrourus, showed that light was the only factor of those investigated that was obligatory for hatching. It was found that eggs which had not been desiccated hatched successfully in the presence of absence of adults, while those which had been desiccated showed a block in hatching initially, although this block deteriorated with time and after approximately two months the eggs which had been desiccated showed a similar hatching success to that of the non-desiccated eggs. Exposure of eggs to extremes of heat or cold before incubation did not influence the hatching success of the eggs significantly, but the temperature at which incubation took place was important. The optimal range lay between 14 °C and 20 °C. Eggs hatched and nauplii survived at dissolved oxygen tensions of below 0.5 mg 1^–1     

Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation,Replication, and Persistence

Kaposi''s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.     

Syntheses and Proton Magnetic Resonance Studies at 300 MHz of Two New Bromopentachlorocyclohexanes and Three New Bromotrichlorocyclohexenes

Two diastereomers of 6-bromo-1, 2, 3, 4, 5-pentachlorocyclohexane were synthesized from the dl (36/45)-diastereomer of 3, 4, 5, 6-tetrachlorocyclohexene (α-BTC) and the dl (346/5)-diastereomer (γ-BTC) by several stepwise routes. Both of these new products were shown to have the configuration of lindane by NMR studies at 300 MHz and by the synthetic routes. Three diastereomers of 6-bromo-3, 4, 5-trichlorocyclohexene were also prepared and the configurations determined partly by means of 300 MHz NMR.