Baroreflex sensitivity in obesity: relationship with cardiac autonomic nervous system activity

Objective: The aim of this study was to test the hypothesis that baroreflex sensitivity (BRS), assessed by indirect measurement of aortic pressure, is blunted in obesity. Additionally, the potential effect of cardiac autonomic nervous system (ANS) activity, aortic compliance, and metabolic parameters on BRS of obese subjects was investigated. Research Methods and Procedures: A group of 30 women with BMI >30 kg/m² and a group of 30 controls with BMI <25 kg/m² were examined. BRS was estimated by the sequence technique, cardiac ANS activity by short‐term spectral analysis of heart rate variability (HRV), and aortic compliance by the method of applanation tonometry. Results: BRS was lower in obese women (9.18 ± 3.77 vs. 19.63 ± 9.16 ms/mm Hg, p < 0.001). The median values (interquartile range) of the power of both the high‐frequency and low‐frequency components of the HRV were higher in the lean than in the obese participants [1079.2 (202.7 to 1716.9) vs. 224.1 (72.7 to 539.6) msec², p = 0.001 and 411.8 (199.3 to 798.0) vs. 235.8 (99.4 to 424.5) msec², p = 0.01 respectively]. Low‐to‐high‐frequency ratio values were higher in the obese subjects [0.82 (0.47 to 2.1) vs. 0.57 (0.28 to 0.89), p = 0.02]. Aortic augmentation values were not significantly different between lean and obese subjects. Multivariate analysis demonstrated a significant and independent association between BRS and age (p = 0.003), BMI (p < 0.001), and high‐frequency power of HRV (p < 0.001). These variables explained 72% of the variation of BRS values. Discussion: BRS is severely reduced in obese subjects. BMI, age, and the parasympathetic nervous system activity are the main determinants of BRS. Baroreflex behavior is of clinical relevance because an attenuated BRS represents a negative prognostic factor in cardiovascular diseases, which are common in obesity.     

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.     

Synthesis and biological activity of water-soluble maleimide derivatives of the anticancer drug carboplatin designed as albumin-binding prodrugs

Four platinum (II) complexes (13-16) were synthesized by reacting either [Pt trans-DACH](NO(3))(2) with a 6-maleimidocaproic acid, a 15-maleimido-4,7,10,13-tetroxapentadecanoic acid, and a 6-maleimido-4-oxacaproic ester derivative of cyclobutane-1,1-dicarboxylic acid (CDBA) or [Pt(NH(3))(2)](NO(3))(2) with a 6-maleimido-4-oxacaproic ester derivative of CBDA. Both complexes containing the 6-maleimido-4-oxacaproic ester (15, 16) showed good water solubility (>/=8 mg/mL) and CE experiments revealed rapid binding to human serum albumin and the formation of biadducts with dGMP and dAMP. In the MaTu xenograft model in nude mice, both complexes showed an improved antitumor effect at their maximum tolerated dose (2 x 50 mg/kg carboplatin equivalents) compared to therapy with carboplatin at equimolar dose or at its optimal dose (2 x 75 mg/kg).     

Measurement of chemopreventive efficacy in skin biopsies

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.     

Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.     

Maleimide-oligo(ethylene glycol) derivatives of camptothecin as albumin-binding prodrugs: synthesis and antitumor efficacy

In situ binding of thiol-reactive prodrugs to the cysteine-34 position of circulating albumin is a new approach in drug delivery. Therefore, five maleimide-bearing derivatives of the anticancer drug camptothecin (CPT) were developed as albumin-binding prodrugs. These compounds were synthesized by reacting heterobifunctional cross-linkers based on oligo(ethylene glycols) [3-6 (O-CH(2)-CH(2)) units] bearing a maleimide group on one end and a carboxylic acid group on the other with camptothecin 20-O-glycinate. Incorporating oligo(ethylene glycol) chains into the prodrugs enhanced their water-solubility when compared to the parent compound (up to 27-fold). HPLC studies showed that the prodrugs react almost quantitatively with the cysteine-34 position of endogenous albumin within a few minutes after incubation of the CPT derivatives with human blood plasma. The therapeutic potential of two of the prodrugs was assessed in nude mice bearing a colon xenograft (HT-29). Both albumin-binding derivatives of camptothecin were well-tolerated and showed enhanced antitumor efficacy when compared to CPT.     

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.     

Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples

AIMS: An internal positive control for Cryptosporidium and Giardia monitoring was evaluated for use in routine water monitoring quality control. The control, known as ColorSeed C&G (BTF Pty Ltd, Sydney, Australia), is a suspension containing exactly 100 Cryptosporidium oocysts and 100 Giardia cysts that have been modified by attachment of Texas Red to the cell wall, allowing them to be differentiated from unmodified oocysts and cysts. The control enables recovery efficiencies to be determined for every water sample analysed. METHODS AND RESULTS: A total of 494 water samples were seeded with ColorSeed C&G and with unlabelled Cryptosporidium and Giardia and then analysed. Additionally, the robustness of the ColorSeed labelling was challenged with various chemical treatments. Recoveries were significantly lower for the ColorSeed Texas Red labelled Cryptosporidium and Giardia than recoveries of unlabelled Cryptosporidium and Giardia. However, the differences in recoveries were small. On average ColorSeed Cryptosporidium recoveries were 3.3% lower than unlabelled Cryptosporidium, and ColorSeed Giardia recoveries were 4% lower than unlabelled Giardia. CONCLUSIONS: ColorSeed C&G is suitable for use as an internal positive control for routine monitoring of both treated and raw water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The small differences in recoveries are unlikely to limit the usefulness of ColorSeed C&G as an internal positive control. The ColorSeed labelling was found to be robust after different treatments.     

Genetic and biochemical characterization of MbeA,the relaxase involved in plasmid ColE1 conjugative mobilization

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.