Effects of Age and Parity on Mammary Gland Lesions and Progenitor Cells in the FVB/N-RC Mice

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.     

The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis

Conjugal DNA transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmid-mediated DNA transfer. Previously, we had shown that the secretory apparatus, ESX-1, negatively regulated DNA transfer from the donor strain; ESX-1 donor mutants are hyper-conjugative. Here, we describe a genome-wide transposon mutagenesis screen to isolate recipient mutants. Surprisingly, we find that a majority of insertions map within the esx-1 locus, which encodes the secretory apparatus. Thus, in contrast to its role in donor function, ESX-1 is essential for recipient function; recipient ESX-1 mutants are hypo-conjugative. In addition to esx-1 genes, our screen identifies novel non- esx-1 loci in the M. smegmatis genome that are required for both DNA transfer and ESX-1 activity. DNA transfer therefore provides a simple molecular genetic assay to characterize ESX-1, which, in Mycobacterium tuberculosis , is necessary for full virulence. These findings reinforce the functional intertwining of DNA transfer and ESX-1 secretion, first described in the M. smegmatis donor. Moreover, our observation that ESX-1 has such diametrically opposed effects on transfer in the donor and recipient, forces us to consider how proteins secreted by the ESX-1 apparatus can function so as to modulate two seemingly disparate processes, M. smegmatis DNA transfer and M. tuberculosis virulence.     

Spectroscopic characterization of the light-harvesting complex of Rhodospirillum rubrum and its structural subunit

The spectroscopic properties of the light-harvesting complex of Rhodospirillum rubrum, B873, and a detergent-isolated subunit form, B820, are presented. Absorption and circular dichroism spectra suggest excitonically interacting bacteriochlorophyll alpha (BChl alpha) molecules give B820 its unique spectroscopic properties. Resonance Raman results indicate that BCHl alpha is 5-coordinate in both B820 and B873 but that the interactions with the BChl C2 acetyl in B820 and B873 are different. The reactivity of BChl alpha in B820 in light and oxygen, or NaBH4, suggests that it is exposed to detergent and the aqueous environment. Excited-state lifetimes of the completely dissociated 777-nm-absorbing form [1.98 ns in 4.5% octyl glucoside (OG)], the intermediate subunit B820 (0.72 ns in 0.8% OG), and the in vivo like reassociated B873 (0.39 ns in 0.3% OG) were measured by single-photon counting. The fluorescence decays were exponential when emission was detected at wavelengths longer than 864 nm. An in vivo like B873 complex, as judged by its spectroscopic properties, can be formed from B820 without the presence of a reaction center.     

A novel metabolic form of the 32 kDa-D1 protein in the grana-localized reaction center of photosystem II

Two forms of the 32 kDa-D1 reaction center protein of photosystem II (PSII), having slightly different mobilities on denaturing polyacrylamide gels, have been resolved in Spirodela oligorrhiza, Glycine max L., Gossypium hirsutum L., Triticum aestivum L., and Zea mays L. The protein band with faster mobility is identified as the 32 kDa-D1 protein, and the less mobile band as a novel form, designated 32*. The two forms are structurally similar based on immunological and partial proteolytic tests. 32* is associated exclusively with the grana and is present in the PSII reaction center. Temporally, 32* appears several hours after the translocation of newly synthesized and processed 32 kDa-D1 protein from the stroma lamellae to the grana. Formation of the 32* is strictly light-dependent under physiological light intensities and correlates with a reciprocal loss of the 32-kDa form. Light induced formation of 32* is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is not coupled to linear electron transport.     

In vitro activation of neuraminidase in the beta-galactosidase-neuraminidase-protective protein complex by cathepsin C

Neuraminidase can be activated by incubation of crude glycoprotein fractions at acidic pH for 90 minutes at physiological temperature. This activation is inhibited by leupeptin. Incubation of the purified neuraminidase-beta-galactosidase-protective protein complex under the same conditions used for crude glycoprotein fractions did not lead to enhanced neuraminidase activity, but incubation in the presence of exogenous Cathepsin C at 4 degrees C resulted in marked enhancement of neuraminidase activity. This activation was again inhibited by leupeptin. Cathepsin D treatment resulted in destruction of neuraminidase under the same conditions and this effect was again inhibited by leupeptin. beta-galactosidase in crude glycoprotein fractions and in the complex was resistant to both Cathepsin C and D, while homogeneous beta-galactosidase was inactivated by these enzymes. We suggest that in vitro activation of neuraminidase may mimic the in vivo intralysosomal conversion of the neuraminidase precursor into the mature form of the enzyme.     

Design,synthesis and evaluation of potent and selective inhibitors of mono-(ADP-ribosyl)transferases PARP10 and PARP14

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with?～15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.