Microbial Hydroxylation of 1,4-Cineole

Microorganisms were examined for their potential to hydroxylate the oxygenated monoterpene 1,4-cineole. Using gas chromatography and thin-layer chromatography, screening experiments revealed that hydroxylation at position 2 was the most commonly observed microbial transformation reaction. In most microorganisms, the predominant alcohol metabolite was the 2-endo-alcohol isomer. Preparative-scale incubations were conducted in order to isolate and characterize microbial transformation products by comparison of proton nuclear magnetic resonance, mass spectrometry, and chromatography profiles with those of cineole standards. Streptomyces griseus yielded 8-hydroxy-1,4-cineole as the major hydroxylation product together with 2-exo- and 2-endo-hydroxy-1,4-cineoles.     

Institutional Delivery and Satisfaction among Indigenous and Poor Women in Guatemala,Mexico, and Panama

Indigenous women in Mesoamerica experience disproportionately high maternal mortality rates and are less likely to have institutional deliveries. Identifying correlates of institutional delivery, and satisfaction with institutional deliveries, may help improve facility utilization and health outcomes in this population. We used baseline surveys from the Salud Mesoamérica Initiative to analyze data from 10,895 indigenous and non-indigenous women in Guatemala and Mexico (Chiapas State) and indigenous women in Panama. We created multivariable Poisson regression models for indigenous (Guatemala, Mexico, Panama) and non-indigenous (Guatemala, Mexico) women to identify correlates of institutional delivery and satisfaction. Compared to their non-indigenous peers, indigenous women were substantially less likely to have an institutional delivery (15.2% vs. 41.5% in Guatemala (P<0.001), 29.1% vs. 73.9% in Mexico (P<0.001), and 70.3% among indigenous Panamanian women). Indigenous women who had at least one antenatal care visit were more than 90% more likely to have an institutional delivery (adjusted risk ratio (aRR) = 1.94, 95% confidence interval (CI): 1.44–2.61), compared to those who had no visits. Indigenous women who were advised to give birth in a health facility (aRR = 1.46, 95% CI: 1.18–1.81), primiparous (aRR = 1.44, 95% CI: 1.24–1.68), informed that she should have a Caesarean section (aRR = 1.41, 95% CI: 1.21–1.63), and had a secondary or higher level of education (aRR = 1.36, 95% CI: 1.04–1.79) also had substantially higher likelihoods of institutional delivery. Satisfaction among indigenous women was associated with being able to be accompanied by a community health worker (aRR = 1.15, 95% CI: 1.05–1.26) and facility staff speaking an indigenous language (aRR = 1.10, 95% CI: 1.02–1.19). Additional effort should be exerted to increase utilization of birthing facilities by indigenous and poor women in the region. Improving access to antenatal care and opportunities for higher-level education may increase institutional delivery rates, and providing culturally adapted services may improve satisfaction.     

Degree of modification of Ro60 by the lipid peroxidation by-product 4-hydroxy-2-nonenal may differentially induce Sjögren syndrome or systemic lupus erythematosus in BALB/c mice

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.     

Activity of dermaseptin K4-S4 against foodborne pathogens

Dermaseptin S4 and its substituted derivative K(4)-S4 were investigated against various food-related pathogenic bacteria in culture media. K(4)-S4, but not the native peptide displayed significant growth inhibitory activity against all bacteria tested. Next, activity of K(4)-S4 against Escherichia coli O157:H7 was defined in terms of milieu dependencies. Salt-dependent kinetic studies in growth medium indicated that the peptide's antibacterial activity is maintained at fairly high (up to 600mM) NaCl concentrations but inhibited at higher concentrations. Similarly, antibacterial activity was reduced at high but not low pH conditions. Importantly, antibacterial activity was significantly maintained at temperatures lower than 37 degrees C and significantly enhanced at 42 degrees C. With respect to bactericidal kinetics, negative cultures were obtained in LB as well as in commercial apple juice, respectively, within 1 and 2h treatment, at twice the minimal inhibitory concentration (MIC) value. Overall, the data collected is indicative of a certain interest for dermaseptin derivatives as potential food preservatives.     

Non-linear heteroscedastic regression model for determination of methotrexate in human plasma by high-performance liquid chromatography

Generalized least squares regression with variance function estimation was used to derive the calibration function for measurement of methotrexate plasma concentration and its results were compared with weighted least squares regression by usual weight factors and also with that of ordinary least squares method. In the calibration curve range of 0.05 to 100 microM, both heteroscedasticity and non-linearity were present therefore ordinary least squares linear regression methods could result in large errors in the calculation of methotrexate concentration. Generalized least squares regression with variance function estimation worked better than both the weighted regression with the usual weight factors and ordinary least squares regression and gave better estimates for methotrexate concentration.     

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.     

Prostate-cancer-targeted N-(2-hydroxypropyl)methacrylamide copolymer/docetaxel conjugates

Biodistribution, pharmacokinetics, and efficacy of prostate-cancer-targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4-2 xenografts. PSMA-specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7-fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates.     

The total peroxyl radical trapping potential in serum - an assay to define the stage of atherosclerosis

Lipid peroxides were identified among the factors that contribute to the atherosclerotic plaque formation in the arterial wall. We hypothesised that a correlation may exist between the content of antioxidant constituents in the serum and the gravity of atherosclerosis. To this purpose, we have determined the serum total peroxyl radical- trapping potential (TRAP), which is the combined capacity of all antioxidants to neutralize free radicals in serum and followed its variation in hyperlipemic animals in correlation with the stage of atherosclerosis. In addition, we compared TRAP values in the sera of coronary heart disease (CHD) patients, with or without type II diabetes mellitus. Results showed that after 18 weeks of hyperlipemic diet, the mean TRAP values measured in sera isolated from hyperlipemic hamsters exhibited an about 44% decrease, in good agreement with the increase of serum cholesterol and triglycerides. In the 3 groups of CHD patients, TRAP values decreased with about 10% in sera of stable angina patients, 20% in unstable patients, as compared with normal subjects. The lowest TRAP values were detected in the sera of patients with acute myocardial infarction. The results obtained for different experimental animals and for CHD patients sera indicate that the TRAP method, as adapted in our laboratory, is a reliable and reproducible assay, fit to be used in clinical studies as an ex vivo measurable parameter that correlates with the stage of the atherosclerosis.     

Role of PTPα in the Destruction of Periodontal Connective Tissues

IL-1β contributes to connective tissue destruction in part by up-regulating stromelysin-1 (MMP-3), which in fibroblasts is a focal adhesion-dependent process. Protein tyrosine phosphatase-α (PTPα) is enriched in and regulates the formation of focal adhesions, but the role of PTPα in connective tissue destruction is not defined. We first examined destruction of periodontal connective tissues in adult PTPα^+/+ and PTPα^−/− mice subjected to ligature-induced periodontitis, which increases the levels of multiple cytokines, including IL-1β. Three weeks after ligation, maxillae were processed for morphometry, micro-computed tomography and histomorphometry. Compared with unligated controls, there was ∼1.5–3 times greater bone loss as well as 3-fold reduction of the thickness of the gingival lamina propria and 20-fold reduction of the amount of collagen fibers in WT than PTPα^−/− mice. Immunohistochemical staining of periodontal tissue showed elevated expression of MMP-3 at ligated sites. Second, to examine mechanisms by which PTPα may regulate matrix degradation, human MMP arrays were used to screen conditioned media from human gingival fibroblasts treated with vehicle, IL-1β or TNFα. Although MMP-3 was upregulated by both cytokines, only IL-1β stimulated ERK activation in human gingival fibroblasts plated on fibronectin. TIRF microscopy and immunoblotting analyses of cells depleted of PTPα activity with the use of various mutated constructs or with siRNA or PTPα^KO and matched wild type fibroblasts were plated on fibronectin to enable focal adhesion formation and stimulated with IL-1β. These data showed that the catalytic and adaptor functions of PTPα were required for IL-1β-induced focal adhesion formation, ERK activation and MMP-3 release. We conclude that inflammation-induced connective tissue degradation involving fibroblasts requires functionally active PTPα and in part is mediated by IL-1β signaling through focal adhesions.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.