In the present study expression of estrogen receptor subtype -alpha (ERalpha) and -beta (ERbeta) in the cerebral cortex, cerebellum, and olfactory bulb was investigated and compared between neonatal ...(1 to approximately 3-days-old) and adult (250 to approximately 350 g) rats, using reverse transcription-polymerase chain reaction (RT-PCR). No ERalpha transcripts were detectable in the adult cerebellum and olfactory bulb, whereas very weak expression of ERalpha was present in the adult cerebral cortex. No significant difference in ERbeta transcripts was detectable between the neonatal and adult rats. While transcripts for both ER subtypes were co-expressed in these brain areas of neonatal rats, although ERalpha expression was significantly weaker than ERbeta. Even in the cerebral cortex known to contain both ER subtypes in adult rats, ERalpha transcripts in neonatal rats were much higher than in adult. These observations provide evidence for the existence of different expression patterns of ERalpha/ERbeta transcripts in these three brain areas between the neonatal and adult rats, suggesting that each ER subtype may play a distinct role in the regulation of differentiation, development, and functions of the brain by estrogen.
APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other ...effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.
Epothilones are a new class of natural products that bind to tubulin and prevent the depolymerization of microtubules, although they have no structural similarity to paclitaxel. Taxanes are only ...marginally effective in the treatment of disseminated prostate cancer, although they may have useful activity when administered in combination with estramustine. Unlike paclitaxel, epothilones are not substrates for P-glycoprotein and are active in multidrug resistant cells. Epothilones A and B (EA, EB) have recently been synthesized in toto. In this report, we examine the effects of synthetic epothilones and their desoxy derivatives, as well as paclitaxel, on prostate cancer cell lines. EB was the most active of these compounds in tissue culture (IC(50): 50-75 pM), four to ten-fold more potent than paclitaxel. EA and the desoxyderivatives of EA and EB (dEA, dEB) were also active, but less potent than EB. Each of these compounds causes mitotic block followed by apoptotic cell death. The relative potencies for cell cycle arrest and cytotoxicity directly correlate with the ability of the drugs to bind microtubules, stabilize mitotic spindles and induce the formation of interphase microtubule bundles. Therefore, synthetic epothilones are potent inhibitors of prostate cancer cell lines and work in a fashion similar to paclitaxel. Recently, we showed that farnesyl transferase inhibitors sensitize tumor cells to paclitaxel-induced mitotic arrest. We now have extended these observations to show that paclitaxel and the epothilones synergize with FTI to arrest the growth of prostate cancer cells. Moreover, this occurs in DU145, a cell line that is not particularly sensitive to the FTI. The combination of FTI and epothilone represent a new potential clinical strategy for the treatment of advanced prostatic cancer.
There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic ...infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-α, and TGF-β offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene-deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-α, TGF-β, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.
Background Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis. The R2 subunit of
Escherichia coli RNR is a homodimer containing ...one dinuclear iron centre per monomer. A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen. To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2.
Results The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211→ Ala) have been determined at 1.7 å and 2.2 å resolution, respectively. The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2. In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging
μ-oxo group and two water molecules are lost. In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen. The structure of the mutant Ser211→ Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein.
Conclusions Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions. In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen. The Ser211→ Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.
Our previous studies demonstrated that intrathecal lidocaine treatment could produce prolonged reversal of established hyperalgesia or allodynia, both induced by chronic constriction injury. Indeed, ...intrathecal lidocaine treatment remarkably suppressed the activation of p38 mitogen-activated protein kinase (MAPK) in hyperactive microglia. In the present study we suggest that lidocaine may act directly on the microglia and attenuate the release of cytokines.
We assessed the influence of lidocaine on the levels of phospho-p38 MAPK, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and intracellular calcium triggered by extracellular adenosine triphosphate (ATP) in cultured rat microglia. Our experimental methods included Western blot, real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and calcium imaging.
We found that lidocaine (in a dose-dependent manner) significantly attenuated p38 MAPK activation triggered by 1 mM ATP, by inhibiting the transcription of 3 cytokine messenger RNAs and causing a decrease in their respective protein concentrations (TNF-alpha, IL-1beta, and IL-6, P < 0.05, vs. the ATP group). SB203580, an antagonist of P38, attenuated ATP-activated elevation in protein levels of TNF-alpha, IL-1beta, and IL-6 in the microglia. The high level of intracellular calcium (Ca(2+)i) that is induced by ATP was decreased by the addition of 10 mM lidocaine (P < 0.05 vs. the ATP group).
These findings indicate that lidocaine can directly act on microglia. Lidocaine, by inhibiting the increase of intracellular calcium, also inhibited p38 MAPK activation and attenuated the production of proinflammatory cytokines (including TNF-alpha, IL-1beta, and IL-6), which were triggered by extracellular ATP in cultured rat microglia.
The present study was designed to testify the hypothesis that arterial baroreflex dysfunction promotes the development of atherosclerosis.
Experiment 1: the baroreflex sensitivity (BRS) was measured ...in 30 Sprague-Dawley (SD) rats in conscious state with a computerized blood pressure monitoring system. Four weeks later, the rats were administered with Vitamin D
3, and fed with the high-cholesterol diet for 8 weeks to induce atherosclerosis. The hearts and aortae were removed for pathological examination. A negative correlation was found between BRS and the scores of coronary (
r
=
−0.464,
P
<
0.01) or aortic atherosclerosis (
r
=
−0.524,
P
<
0.01) in SD rats.
Experiment 2: sinoaortic denervation (SAD) was performed in SD rats. Then atherosclerosis was also induced. The atherosclerosis scores in SAD rats were significantly higher than those in sham-operated rats (aortic score: 1.50
±
0.41 versus 1.10
±
0.39,
P
<
0.05; coronary score: 1.70
±
0.35 versus 1.25
±
0.54,
P
<
0.05). Using immunohistochemistry and Western blotting methods, it was found that the expressions of C-reactive protein, intercellular adhesion molecule-1 and vascular-cell adhesion molecule-1 in coronary artery and aorta were increased in SAD rats compared with sham-operated rats. These results indicate that arterial baroreflex dysfunction promotes the development of atherosclerosis in rats, and that inflammation may be involved in this process.
Background: Natural killer and natural killer‐like T‐cell lymphomas presenting in the skin usually demonstrate aggressive behavior, an angiocentric distribution and a characteristic immunophenotype. ...In contrast, primary cutaneous CD30+ lymphoproliferative disorders form a heterogeneous spectrum including anaplastic large cell lymphomas, the majority of which display a good prognosis. Lymphomas with co‐expression of CD56 and CD30 are extremely rare and the significance of this co‐expression is unknown.
Methods: Seven retrospectively identified cases of lymphomas with co‐expression of CD56 and CD30 presenting in the skin comprise this study. Immunohistochemistry, in situ hybridization for Epstein‐Barr virus and T‐cell receptor gene rearrangement studies were performed on paraffin sections.
Results: This subset of cutaneous lymphomas showed a variable clinical course that ranged from resolution without treatment, treatment‐failure and recurrence, to death from disease. Histologic, immunophenotypic and molecular studies were of limited utility in predicting prognosis.
Conclusions: Cutaneous lymphomas co‐expressing CD56 and CD30 share many clinicopathologic features with natural killer and natural killer‐like T‐cell lymphomas or anaplastic large cell lymphomas, two entities with widely disparate clinical behavior. It is important to recognize that these lymphomas may behave more aggressively than primary cutaneous anaplastic large cell lymphomas do. Longer follow‐up and further investigations on larger numbers of cases are necessary to fully characterize this rare subset of cutaneous lymphomas.
The mda-7 gene (approved gene symbol IL24) is a novel tumor suppressor gene with tumor-apoptotic and immune-activating properties. We completed a Phase I dose-escalation clinical trial, in which a ...nonreplicating adenoviral construct expressing the mda-7 transgene (INGN 241; Ad-mda7) was administered intratumorally to 22 patients with advanced cancer. Excised tumors were evaluated for vector-specific DNA and RNA, transgenic MDA-7 expression, and biological effects. Successful gene transfer as assessed by DNA- and RT-PCR was demonstrated in 100% of patients evaluated. DNA analyses demonstrated a dose-dependent penetration of INGN 241 (up to 4 × 108 copies/μg DNA at the 2 × 1012 vp dose). A parallel distribution of vector DNA, vector RNA, MDA-7 protein expression, and apoptosis induction was observed in all tumors, with signals decreasing with distance away from the injection site. Additional evidence for bioactivity of INGN 241 was illustrated via regulation of the MDA-7 target genes β-catenin, iNOS, and CD31. Transient increases (up to 20-fold) of serum IL-6, IL-10, and TNF-α were observed. Significantly higher elevations of IL-6 and TNF-α were observed in patients who responded clinically to INGN 241. Patients also showed marked increases of CD3+CD8+ T cells posttreatment, suggesting that INGN 241 increased systemic TH1 cytokine production and mobilized CD8+ T cells. Intratumoral delivery of INGN 241 induced apoptosis in a large volume of tumor and elicited tumor-regulatory and immune-activating events that are consistent with the preclinical features of MDA-7/IL-24.
To review the best-corrected visual acuity, ulcer size, microbiological profile and morbidity of contact lens-related microbial keratitis with and without prior topical steroid use.
Retrospective ...case review of admitted cases of contact lens-related microbial keratitis in a tertiary hospital. Data pertaining to demographics, pre-admission treatment with or without topical steroids, ulcer size, duration of admission, Gram stain and culture results as well as the final best-corrected visual acuity were recorded. Patients are classified into 3 groups: Group 1 received no treatment prior to presentation, Group 2 received topical antibiotics only from their general practitioners and Group 3 prescribed both topical antibiotics and steroids.
Forty-six cases were enrolled in the study, 41.3% had prior topical steroids (all dexamethasone) in combination with antibiotics. None of them had topical steroids alone. Large ulcers were associated with steroid use, odds ratio = 7.74 95% confidence interval (CI), 1.18-50.56 and positivity of Gram stains odds ratio = 7.74 95% CI, 1.18-50.56 whereas loss of more than 2 Snellen lines was associated with Pseudomonas aeruginosa infection, odds ratios of 21.70 95% CI,2.09-225.03 and presence of central ulcer, 13.51 95% CI, 2.33-78.3. Prior topical steroid use was associated with longer duration of symptoms prior to admission but not duration of stay or surgical intervention.
Patients with prior topical combined antibiotics-steroids present slightly later and with larger ulcers. However, the duration of stay, final visual acuity, treatment failure and complication rates were not statistically different from the non-treated group. This might be due to 1) early presentation and therefore early treatment of contact lens-related microbial keratitis and 2) the short duration of use of combined antibiotic-steroid eye drops.
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