Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver.

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.     

Solid-phase synthesis, bioconjugation, and toxicology of novel cationic oligopeptoids for cellular drug delivery

For many therapeutic applications, it has become more and more important to find synthetic compounds that have the ability to transport a variety of drugs and cargo molecules into cells and tissues. Like arginine-rich cell-penetrating peptides (CPPs), it is already known that peptide mimetics such as beta-peptides and peptoids can also express a transport function. In this study, ten fluorophore-labeled chiral and achiral peptoids with different backbone lengths and side chains as well as three peptoids coupled to a therapeutically active porphyrin moiety were prepared using a highly modular solid-phase synthesis (SPP) approach. To compare the structural determinants with the cellular uptake efficiency, all peptoids were analyzed by live cell imaging. All cells show an even vesicular distribution of the internalized peptoids, also revealing that a vesicular escape into the cytosol was stronger for peptoids with longer backbones. Moreover, the uptake efficiency correlated with both the incubation time and the given concentration. Toxicology tests and uptake experiments with porphyrin-coupled peptoids indicate their suitability for application as robust and readily available drug delivery systems or intracellular probes.     

The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.     

Enzymatic resolution and base pairing properties of D- and L-cyclohexenyl nucleic acids (CeNA)

An enzymatic transesterification reaction afforded large scale resolution of the cyclohexenol precursor needed for preparation of both series of CeNA building blocks. CeNA oligos of "D-like" chirality display a strong and selective interaction with RNA, while preserving RNase H activity, and therefore have potential as antisense constructs. CeNAs of opposite chirality form a self-pairing system on their own.     

Biomarkers of exposure and potential harm in adult smokers of 3-7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers.

The paper reports levels of 24-h urine nicotine and five of its major metabolites (expressed as nicotine-equivalents) and blood carboxyhaemoglobin as biomarkers of exposure to particulate- and gas-phase cigarette smoke, respectively, from an exploratory pilot study of adult smokers of 3.0-6.9 mg tar delivery (Federal Trade Commission (FTC) method) cigarettes. On multiple occasions over 6 weeks, blood high-sensitivity C-reactive protein (hs-CRP), fibrinogen, HDL- and LDL-cholesterol, and 24-h urine 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TxB2) were also evaluated as biomarkers of potential harm. All the biomarkers examined, except for LDL-cholesterol, discriminated with high sensitivity and specificity between adult smokers and non-smokers overall. Except for HDL-cholesterol, all biomarker medians were greater in adult smokers than in non-smokers: urine nicotine-equivalents 64.514 versus < 0.034 nmol mg-1 creatinine (p<0.001), carboxyhaemoglobin 4.0 versus 0.4% saturation (p<0.001), hs-CRP 0.27 versus 0.12 mg dl-1 (p=0.05), fibrinogen 292 versus 248 mg dl-1 (p<0.001), HDL-cholesterol 46 versus 53 mg dl-1 (p=0.003), LDL-cholesterol 119 versus 109 mg dl-1 (p=0.18), urine 8-epi-PGF2alpha 1935 versus 1034 pg mg-1 creatinine (p<0.001) and urine 11-dehydro-TxB2 973 versus 710 pg mg-1 creatinine (p<0.001). All the biomarkers of exposure and most of the biomarkers of potential harm showed no time of sampling (by visit week) effect.     

The effects of local perfusion of DAMGO on extracellular GABA and glutamate concentrations in the rostral ventromedial medulla

Electrophysiological data suggest an involvement of rostral ventromedial medulla (RVM) GABA and glutamate (GLU) neurons in morphine analgesia. Direct evidence that extracellular concentrations of GABA or GLU are altered in response to mu opioid receptor (MOP-R) activation is, however, lacking. We used in vivo microdialysis to investigate this issue. Basal GABA overflow increased in response to intra-RVM perfusion of KCl (60 mmol/L). Reverse microdialysis of the MOP-R agonist d -Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) (20–500 μmol/L) produced a concentration-dependent decrease of RVM GABA overflow. Behavioral testing revealed that concentrations that decreased GABA levels increased thermal withdrawal thresholds. A lower agonist concentration that did not increase GABA failed to alter thermal thresholds. DAMGO did not alter GLU concentrations. However, KCl also failed to modify GLU release. Since rapid, transporter-mediated uptake may mask the detection of changes in GLU release, the selective excitatory amino acid transporter inhibitor pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mmol/L) was added to the perfusion medium for subsequent studies. tPDC increased GLU concentrations, confirming transport inhibition. KCl increased GLU dialysate levels in the presence of tPDC, demonstrating that transport inhibition permits detection of depolarization-evoked GLU overflow. In the presence of tPDC, DAMGO increased GLU overflow in a concentration-dependent manner. These data demonstrate that MOP-R activation decreases GABA and increases GLU release in the RVM. We hypothesize that the opposing effects of MOP-R on GLU and GABA transmission contribute to opiate antinociception.     

Hexitol Nucleic Acids (HNA): Synthesis and Properties

Abstract

While improved alkylation procedures have been worked out for the coupling of purine bases to the anhydrohexitol ring using sulphonate activating groups on the anhydrohexitol ring, the Mitsunobu reaction seems to be the method of choice for synthesis of the pyrimidine analogues. In a mixed sequence context, the anhydrohexitol oligonucleotides still display strong and very selective basepairing properties, with a strong preference for RNA as the complement.     

Microplitis croceipes teratocytes: in vitro culture and biological activity of teratocyte secreted protein

Teratocytes originate from the dissociation of the extraembryonic serosal membrane in some Braconidae and Scelionidae. Methods used to culture teratocytes in vitro are described and the yield of teratocyte secreted proteins (TSP) was measured. Although 90% are viable after 6 days, in vitro teratocytes reached only half the diameter (32&mgr;m) of the same age teratocytes obtained in vivo. Teratocytes cultured in vitro secrete as much as 0.7&mgr;g of protein per day per larval equivalent ( approximately 900 cells). Presence of parasitoid larvae enhanced teratocyte viability while periodic exchange of medium did not. However, medium exchange significantly increased the total amount of protein secreted. Size and viability were improved with the addition of 10% FBS to the Ex-cell 400 culture medium. Non-denaturing PAGE showed at least 15 proteins with molecular sizes estimated to be between 24 to 347kDa in medium containing teratocytes. An in vitro fat body assay was developed to measure the effect of TSP on protein synthesis and juvenile hormone esterase (JHE) activity. Crude TSP inhibited in vitro incorporation of [(35)S]-methionine into protein synthesized by the fat body. The amount of JHE released from in vitro fat body treated with crude TSP was significantly less than controls, most likely caused by the inhibition of general protein synthesis. The active fraction of TSP passed through a 30kDa molecular weight cutoff filter but was retained by a 3kDa filter. SDS-PAGE revealed four proteins with molecular weights between 8 and 20kDa not present in control medium incubated without teratocytes.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.