In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

Thiazole-substituted benzenesulfonamides as inhibitors of 12 human carbonic anhydrases

Four series of para or meta - substituted thiazolylbenzenesulfonamides bearing Cl substituents were designed, synthesized, and evaluated as inhibitors of all 12 catalytically active recombinant human carbonic anhydrase (CA) isoforms. Observed affinities were determined by the fluorescent thermal shift assay and the intrinsic affinities were calculated based on the fractions of binding-ready deprotonated sulfonamide and CA bearing protonated hydroxide bound to the catalytic Zn(II) in the active site. Several compounds exhibited selectivity towards CA IX, an anticancer target. Intrinsic affinities reached 30 pM, while the observed affinities - 70 nM. The structure-intrinsic affinity relationship map of the compounds showed the energetic contributions of the thiazole ring and its substituents.     

Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC₅₀ values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC₅₀ > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.     

FlhF regulates the number and configuration of periplasmic flagella in Borrelia burgdorferi

The Lyme disease bacterium Borrelia burgdorferi has 7–11 periplasmic flagella (PF) that arise from the cell poles and extend toward the midcell as a flat-ribbon, which is distinct from other bacteria. FlhF, a signal recognition particle (SRP)-like GTPase, has been found to regulate the flagellar number and polarity; however, its role in B. burgdorferi remains unknown. B. burgdorferi has an FlhF homolog (BB0270). Structural and biochemical analyses show that BB0270 has a similar structure and enzymatic activity as its counterparts from other bacteria. Genetics and cryo-electron tomography studies reveal that deletion of BB0270 leads to mutant cells that have less PF (4 ± 2 PF per cell tip) and fail to form a flat-ribbon, indicative of a role of BB0270 in the control of PF number and configuration. Mechanistically, we demonstrate that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS-ring protein FliF. Our study not only provides the detailed characterizations of BB0270 and its profound impacts on flagellar assembly, morphology and motility in B. burgdorferi, but also unveils mechanistic insights into how spirochetes control their unique flagellar patterns.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Effects of amiprilose hydrochloride on the components of human skin equivalents

Summary Amiprilose hydrochloride has been shown to inhibit the proliferation of a number of hyperproliferative cell types including psoriatic skin cells. In the present study, the effects of amiprilose hydrochloride on human tissue equivalents were examined by incubating a) dermal equivalents, b) skin equivalents in the process of epidermalization, and c) mature skin equivalents, with varying concentrations of the drug. In all three models amiprilose hydrochloride concentrations of 0.1% (wt/vol) and lower were not toxic to fibroblasts and keratinocytes and did not interfere with the differentiation of the skin equivalent and the developing skin equivalent. When tested in dermal equivalents, concentrations of amiprilose hydrochloride between 0.1 and 0.5% resulted in changes in fibroblast morphology with development of large intracellular vacuoles, and concentrations greater than 5% were toxic. In mature skin equivalents, in addition to changes in fibroblast morphology, amiprilose hydrochloride in concentrations of 1 to 10% affected the epidermis. When 0.5% amiprilose hydrochloride was present in the developing skin equivalent during differentiation, the epidermal keratinocytes were also affected. Thus the morphology of basal keratinocytes was modified, the differentiation was incomplete, and the dermalepidermal attachment was compromised. These studies suggest the possibility of an extracellular mechanism of action of amiprilose hydrochloride and delineate acceptable dosage ranges for the potential drug. Supported in part by research grant AG01274 from the National Institutes of Health, Bethesda, MD, The R. A. Welch Foundation (B0502), The Texas Advanced Technology and Research Program (Wound Healing and Aging no. 2147), and Greenwich Pharmaceuticals, Inc. R. W. G. is the recipient of a MERIT award from the National Institute on Aging, Bethesda, MD.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis and in vitro antiproliferative evaluation of novel N-alkylated 6-isobutyl- and propyl pyrimidine derivatives

A series of novel N-alkylated C-6-isobutyl- or -propyl pyrimidine derivatives were synthesized and their antiproliferative effect was evaluated on a panel of tumor cell lines including leukemia cell line K562 and normal diploid human fibroblasts. N-methoxymethylated 5-methylpyrimidin-2,4-dione with di(benzyloxy)isobutyl at C-6 (14b) showed the strongest effect on the cell growth at micromolar concentrations. Mechanisms of action for the lipophilic compound 14b predicted in silico, pointed to its anticancer and antimetastatic potential exerted through inhibition of DNA or RNA polymerases and adhesion molecules. The latter mechanism has been supported in vitro for adherent tumor cell lines.