Nanoscale enzyme inhibitors: Fullerenes inhibit carbonic anhydrase by occluding the active site entrance

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar—low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1 nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.     

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf–MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)–ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.     

Effects of ovariectomy and tamoxifen on rat bone tissue: histomorphometric and histopathologic study

OBJECTIVE: To investigate possible detrimental effects on bone tissue induced by ovariectomy and tamoxifen (TMX) using bone densitometry and histomorphologic analysis. STUDY DESIGN: Twenty-four rats were allocated into 4 groups: group 1, intact normal rats (n = 6); group 2, ovariectomized rats (n = 6); group 3, normal female rats that received 1 mg/kg/day TMX dissolved in dimethyl sulfoxide (DMSO) for 2 months (n = 6); group 4, normal female rats that received DMSO for the same duration and with a volume equal to that of TMX (n = 6). Results of histomorphometric analysis for trabecular thickness, number of osteoblasts and osteoclasts, trabecular number, and area and cortical thickness were compared. RESULTS: No significant effects of ovariectomy on femoral or lumbar bone mineral density (BMD) were found. In the TMX group, the value of femoral BMD increased significantly compared to control group cellular and pathologic changes. TMX caused significant decrease in osteoblasts compared to the control group. CONCLUSION: TMX has a positive effect on inorganic bone tissue, but a negative effect on number of osteoblasts and osteoclasts. Future studies investigating estrogenic and antiestrogenic effects of TMX should include cellular parameters related to proliferation using histopathologic and histomorphometric analyses.     

A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.     

Age-dependent renal cortical microvascular loss in female mice

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18-22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20-200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm2, P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm2, P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200-500 μm) decreased only in aged sham (P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.