Point and trend accuracy of a continuous intravenous microdialysis-based glucose-monitoring device in critically ill patients: a prospective study.

BACKGROUND: Microdialysis is a well-established technology that can be used for continuous blood glucose monitoring. We determined point and trend accuracy, and reliability of a microdialysis-based continuous blood glucose-monitoring device (EIRUS(®)) in critically ill patients. METHODS: Prospective study involving patients with an expected intensive care unit stay of ≥48 h. Every 15 min, device readings were compared with blood glucose values measured in arterial blood during blocks of 8 h per day for a maximum of 3 days. The Clarke error grid, Bland-Altman plot, mean absolute relative difference and glucose prediction error analysis were used to express point accuracy and the rate error grid to express trend accuracy. Reliability testing included aspects of the device and the external sensor, and the special central venous catheter (CVC) with a semipermeable membrane for use with this device. RESULTS: We collected 594 paired values in 12 patients (65 [26-80; 8-97] (median [IQR; total range]) paired values per patient). Point accuracy: 93.6 % of paired values were in zone A of the Clarke error grid, 6.4 % were in zone B; bias was 4.1 mg/dL with an upper limit of agreement of 28.6 mg/dL and a lower level of agreement of -20.5 mg/dL in the Bland-Altman analysis; 93.6 % of the values ≥75 mg/dL were within 20 % of the reference values in the glucose prediction error analysis; the mean absolute relative difference was 7.5 %. Trend accuracy: 96.4 % of the paired values were in zone A, and 3.3 and 0.3 % were in zone B and zone C of the rate error grid. Reliability: out of 16 sensors, 4 had to be replaced prematurely; out of 12 CVCs, two malfunctioned (one after unintentional flushing by unsupervised nurses of the ports connected to the internal microdialysis chamber, causing rupture of the semipermeable membrane; one for an unknown reason). Device start-up time was 58 [56-67] min; availability of real-time data was 100 % of the connection time. CONCLUSIONS: In this study in critically ill patients who had no hypoglycemic episodes and a limited number of hyperglycemic excursions, point accuracy of the device was moderate to good. Trend accuracy was very good. The device had no downtimes, but 4 out of 16 external sensors and 2 out of 12 CVCs had practical problems.

Role of the beam profile and sample geometry in the bonded-disk method on the shrinkage kinetics of two dental resins.

OBJECTIVE: To investigate the time evolution of the two dimensional axial shrinkage field for two dental resins in the bonded disk geometry and further test the bonded-disk method. METHODS: An interferometric technique employing a camera was used to image the 2D axial shrinkage field when polymerizing dental resins in real time both during and after light exposure. Four different beam profiles from two light curing units and three sample geometries were utilized to investigate their roles on the 2D axial shrinkage field. RESULTS: The 2D axial shrinkage field correlates qualitatively with the beam profile shortly after the start of light exposure but takes on distinct shapes caused by the rigidity of the coverslip, beam profile, and the resin viscoelasticity. SIGNIFICANCE: Using the conventional bonded disk geometry and uniform beam profile from a light curing unit, the axial 2D shrinkage field was uniform to within 4% in the central part of bonded disk samples.

Expression analysis of genes involved in TLR2-related signaling pathway: Inflammation and apoptosis after ischemic brain injury.

Toll-like receptor 2 (TLR2) is involved in innate immunity in the brain and in the cascade of events after ischemic stroke. The aim of this study was to get an insight into the expression of genes related to TLR2 signaling pathway and associated with inflammation and apoptosis in the later stages of brain response after ischemic injury. Middle cerebral artery occlusion was performed on both wild-type and TLR2(-/-) mice followed by real-time PCR to measure the relative expression of selected genes. In TLR2(-/-) mice expression of genes involved in proinflammatory response was decreased after cerebral ischemia. Tnf was the most prominent cytokine active in the late phase of recovery. Contrary to proinflammatory genes, the expression of Casp8, as a hallmark of apoptosis, was increased in TLR2(-/-) mice, in particular in the late phase of recovery.

In vitro effects of soy phytoestrogens on rat L6 skeletal muscle cells.

Soy isoflavones display estrogenic activity in humans and animals, and thus are referred to as phytoestrogens. This study was performed to observe the effects of the soy isoflavones genistein, daidzein, and glycitein on cell cultures of rat skeletal muscles. [3H]Thymidine incorporation was used to determine cell proliferation, while protein synthesis and degradation were determined by tracking radiolabeled leucine. For the proliferation studies, insulin, estradiol, genistein, daidzein, or glycitein was supplemented at 0, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, or 20 microM, respectively, or in combinations with final concentrations of 0, 0.1, 1, or 10 microM. Genistein reacted most similarly to estradiol, inhibiting proliferation at > or = 1 microM (P < .001). A combination of phytoestrogens resulted in significant inhibition of cell proliferation, but not to the extent observed with genistein alone. For the protein synthesis and degradation experiments, treatments of 0.1 microM dexamethasone or 1 microM concentrations of insulin, genistein, daidzein, or glycitein were used. Phytoestrogens did not inhibit or stimulate protein degradation or synthesis (P > .05). A one-tailed univariate analysis of variance revealed a trend (P < or = .1) in protein stimulation with genistein and glycitein treatments. These results suggest that the tyrosine kinase inhibiting activity of genistein may be affecting phosphorylation of the mitosis-promoting factor, preventing the advancement of the mitotic cell cycle. In addition, at higher total combined concentrations, daidzein and glycitein may be able to outcompete genistein for receptor sites. These results suggest that soy isoflavones in the diet may potentially modulate normal growth and development in humans and animals that ingest soy-based products.

Effects of Soy-derived diets on plasma and liver lipids, glucose tolerance, and longevity in normal, long-lived and short-lived mice.

We examined the effects of diets based on a low isoflavone or a high isoflavone soy protein isolates in normal, growth-hormone receptor knockout and Ames dwarf, and Prop 1 (df) mice that are hypoinsulinemic, insulin-sensitive, and exceptionally long-lived, as well as in growth hormone transgenic mice that are hyperinsulinemic, insulin-resistant, dyslipidemic, and short-lived. Soybean diets tended to normalize plasma cholesterol levels in dwarf and transgenic mice, while low isoflavone diet reduced plasma triglycerides in most of the examined genotypes. The effects of low isoflavone and high isoflavone diets on the levels of free and esterified cholesterol in the liver were strongly genotype-dependent. Fasting blood glucose levels were reduced and glucose tolerance improved by both low isoflavone and high isoflavone diets in growth hormone-transgenic mice and in their normal siblings. Glucose tolerance was also improved by high-isoflavone diet in growth hormone receptor knockout mice. Lifespan was increased by low isoflavone diet in normal mice from two of the examined stocks. High isoflavone diet increased lifespan in normal animals from one line, but reduced lifespan of normal mice from a different line. We conclude that dietary soy protein intake can improve plasma and hepatic lipid profiles, reduce fasting glucose, enhance capacity for glucose tolerance, and prolong life, but all of these effects are strongly genotype-dependent.

Definition of Soybean Genomic Regions That Control Seed Phytoestrogen Amounts.

Soybean seeds contain large amounts of isoflavones or phytoestrogens such as genistein, daidzein, and glycitein that display biological effects when ingested by humans and animals. In seeds, the total amount, and amount of each type, of isoflavone varies by 5 fold between cultivars and locations. Isoflavone content and quality are one key to the biological effects of soy foods, dietary supplements, and nutraceuticals. Previously we had identified 6 loci (QTL) controlling isoflavone content using 150 DNA markers. This study aimed to identify and delimit loci underlying heritable variation in isoflavone content with additional DNA markers. We used a recombinant inbred line (RIL) population ( $n=100$ ) derived from the cross of “Essex” by “Forrest,” two cultivars that contrast for isoflavone content. Seed isoflavone content of each RIL was determined by HPLC and compared against 240 polymorphic microsatellite markers by one-way analysis of variance. Two QTL that underlie seed isoflavone content were newly discovered. The additional markers confirmed and refined the positions of the six QTL already reported. The first new region anchored by the marker BARC-Satt063 was significantly associated with genistein ( $P=0.009$, $Rcirc;2=29.5\%$ ) and daidzein ( $P=0.007$, $Rcirc;2=17.0\%$ ). The region is located on linkage group B2 and derived the beneficial allele from Essex. The second new region defined by the marker BARC-Satt129 was significantly associated with total glycitein ( $P=0.0005$, $Rcirc;2=32.0\%$ ). The region is located on linkage group D1a+Q and also derived the beneficial allele from Essex. Jointly the eight loci can explain the heritable variation in isoflavone content. The loci may be used to stabilize seed isoflavone content by selection and to isolate the underlying genes.

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of /=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

Genomic Regions That Underlie Soybean Seed Isoflavone Content.

Soy products contain isoflavones (genistein, daidzein, and glycitein) that display biological effects when ingested by humans and animals, these effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key to their biological effect. Our objective was to identify loci that underlie isoflavone content in soybean seeds. The study involved 100 recombinant inbred lines (RIL) from the cross of 'Essex' by 'Forrest,' two cultivars that contrast for isoflavone content. Isoflavone content of seeds from each RIL was determined by high performance liquid chromatography (HPLC). The distribution of isoflavone content was continuous and unimodal. The heritability estimates on a line mean basis were 79% for daidzein, 22% for genistein, and 88% for glycitein. Isoflavone content of soybean seeds was compared against 150 polymorphic DNA markers in a one-way analysis of variance. Four genomic regions were found to be significantly associated with the isoflavone content of soybean seeds across both locations and years. Molecular linkage group B1 contained a major QTL underlying glycitein content (P = 0.0001, R(2) = 50.2%), linkage group N contained a QTL for glycitein (P = 0.0033, R(2) = 11.1%) and a QTL for daidzein (P = 0.0023, R(2) = 10.3%) and linkage group A1 contained a QTL for daidzein (P = 0.0081, R(2) = 9.6%). Selection for these chromosomal regions in a marker assisted selection program will allow for the manipulation of amounts and profiles of isoflavones (genistein, daidzein, and glycitein) content of soybean seeds. In addition, tightly linked markers can be used in map based cloning of genes associated with isoflavone content.

Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for anti-gene radiotherapy.

Targeting of radiation damage to specific DNA sequences is the essence of antigene radiotherapy. This technique also provides a tool to study molecular mechanisms of DNA repair on a defined, single radiodamaged site. We achieved such sequence-specific radiodamage by combining the highly localized DNA damage produced by the decay of Auger-electron-emitters such as 125I with the sequence-specific action of triplex-forming oligonucleotides (TFO). TFO complementary to polypurine-polypyrimidine regions of human genes were synthesized and labeled with 125I-dCTP by the primer extension method. 125I-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were isolated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of 125I-TFO-induced double strand breaks (DSB) were assessed by repair of a plasmid possessing a site-specific DSB lesion. Using plasmids containing target polypurine-polypyrimidine tracts, we obtained the fine structure of sequence-specific DNA breaks produced by decay of 125I with single-nucleotide resolution. We showed that the designed 125I-TFO in nanomolar concentrations could bind to and introduce double-strand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. We also showed 125I-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of nearly 80%, with deletions comprising the majority of mutations. The results obtained demonstrate the ability of 125I-TFO to target specific sequences in their natural environment--within eucaryotic nucleus. Repair of 125I-TFO-induced DNA damage should typically result in mutagenic gene inactivation.

A cooperative interaction between soy protein and its isoflavone-enriched fraction lowers hepatic lipids in male obese Zucker rats and reduces blood platelet sensitivity in male Sprague-Dawley rats.

Soy protein diets lower plasma cholesterol in hyperlipoproteinemic human subjects, as well as in animal models. We fed 7-wk-old male obese (fa/fa) and lean Zucker rats a modified AIN-76 diet (20 g protein/kg diet) containing casein (C), low isoflavone soy protein (38 mg isoflavones/kg diet; LI), or high isoflavone soy protein (578 mg isoflavones/kg diet; HI) for 70 d. In obese rats, plasma total cholesterol was 21 and 29% lower in the LI and HI groups, respectively, than in the C group (P:

Absolute structure determination of the highly biologically active bisdehydrodoisynolic acids.

In a project designed to relate the unexpected in vivo and in vitro properties exhibited by (+)- and (-)-bisdehydrodoisynolic acid with their absolute stereochemical structure, an X-ray crystal-structure analysis was undertaken of the highly estrogenic, poorly binding (-) enantiomer. (1) and (13)C NMR spectra are also reported for the first time. The crystal structure shows the cis juxtaposition of the carboxyl and ethyl groups, which are separated by a large torsion angle, and that only the carbon atom holding the carboxyl group is out of the plane in which the remainder of the fused three-ring moiety lies. The crystal structure, which unequivocally characterizes the (-) enantiomer as cis-13(S),14(R) and, implicitly, the (+) enantiomer as cis-13(R),14(S), will be useful in continued studies aimed at explaining the selective estrogen receptor modulation (SERM) of these enantiomers which, in some cases, produces significantly different end-organ effects compared to those of estradiol, in both males and females, affording the promise of a variety of therapeutic and pharmacologic applications.

Gene targeted agents: new opportunities for rational drug development.

In addition to traditional drug development methods designed to modulate the activity of protein targets, knowledge of disease gene DNA sequences provides an opportunity for the highly rational design of therapeutic agents that act at the DNA level through sequence-specific interactions. Among the ligands capable of binding DNA in a precise, sequence-specific manner are oligonucleotides, peptide nucleic acids and polyamides. Various strategies employing these agents to either transiently or permanently alter gene expression have been investigated over the past decade. During the past two to three years, important steps have been taken to illustrate the therapeutic potential of these ligands. Triple-helix (triplex) forming oligonucleotides have been particularly effective DNA-targeting agents with a wide range of applications, including the positive and negative transcriptional regulation of target genes, as well as the controlled delivery of site-specific mutations. This review will focus upon recent advances involving the use of sequence-specific DNA-binding ligands to modify gene expression and/or structure, with particular emphasis on the use of triplex-forming oligonucleotides in these roles.

Ovarian follicle apoptosis in bovine growth hormone transgenic mice.

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.

Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice.

Transgenic mice overexpressing growth hormone (GH) exhibit alterations in the function of the hypothalamic-pituitary-gonadal (HPG) axis and the H-P-adrenal axis. Alterations in the turnover of hypothalamic neurotransmitters, in plasma hormone levels, and in regulation of their release are associated with reproductive deficits, particularly in females. Results reported after publication of our minireview on this subject provided evidence that GH-transgenic mice have increased binding of GH to GH binding proteins in plasma, are hyperinsulinemic and insulin resistant, and have major alterations in energy budgets with increased allocation to growth. Reduced life span and fertility of these animals may be related to insufficient allocation of energy to reproduction and maintenance. Growth hormone resistance induced by transgenic expression of an antagonistic bGH analog or by targeted disruption (knock-out, KO) of the GH receptor (GH-R) gene leads to dramatic suppression of plasma levels of insulin-like growth factor-1 (IGF-1), and dwarf phenotype due to reduced growth and increased adiposity. In both models of GH resistance, there are marked reproductive deficits in females, decline of breeding performance of males, and alterations in the function of the HPG axis. In GH-R-KO females, puberty is delayed, and litter size is reduced. Fetal weights are reduced whereas placental weights are increased, and the weight of newborn pups is reduced despite an increase in the length of gestation. In GH-R-KO males, copulatory behavior and fertility are reduced, plasma PRL is elevated, and responses to luteinizing hormone releasing hormone (LHRH) in vivo and to LH in vitro are suppressed. However, reproductive deficits in GH-R-KO mice are very mild when compared to those described previously in IGF-KO animals. Apparently, the amounts of IGF-1 that may be produced locally in the absence of GH stimulation are sufficient for sexual maturation and fertility in both sexes, whereas quantitative deficits in reproductive function reflect absence of GH-dependent IGF-1 production and other consequences of eliminating GH signaling. The reproduction phenotype of the GH-R-KO mice is also mild when compared to dwarf mice that lack GH, prolactin (PRL), and thyroid stimulating hormone (TSH). This is presumably related to the presence of redundant mechanisms in the stimulatory control of the gonads by the pituitary and the ability of animals capable of producing PRL and TSH to compensate partially for the absence of GH signaling.

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

A soluble neuronal factor alters contractile function of ventricular myocytes without effect on troponin T isoform expression.

OBJECTIVE: The purpose of this investigation was to establish a model system to facilitate identification of the sympathetic neuronal factor(s) that promotes improved contractility in neonatal cardiac myocytes. Conditioned medium from PC12 cells with sympathetic phenotype served as the source of the neuronal factor. METHODS: Contraction frequency, amplitude and velocity of cultured neonatal rat cardiac myocytes were measured by online video analysis. Interventions included in vitro sympathetic innervation, exposure to PC12 conditioned medium, neurotransmitters and antagonists. Metabolic activity was assayed by 2-deoxyglucose uptake. Troponin T isoform expression was analyzed by SDS-polyacrylamide gel electrophoresis. RESULTS: Medium conditioned by neuronal PC12 cells induced contractility changes similar to those induced by in vitro sympathetic innervation. These effects of PC12 conditioned medium and innervation were not suppressed by adrenergic or muscarinic antagonists nor reproduced by neuropeptide Y or somatostatin. Neuronal PC12 conditioned medium but not chromaffin PC12 conditioned medium, increased metabolic activity of the myocytes as detected by [3H]-2-deoxyglucose, indicating that the effect was specific to the neuronal PC12 cells. The in vitro switch of troponin T isoform expression was not altered by exposure to PC12 conditioned medium. CONCLUSIONS: Increased contractile function induced by sympathetic innervation is reproduced by PC12 conditioned medium, but neither is mediated by sympathetic or muscarinic neurotransmitters. Troponin T isoform expression is not related to the contractility changes. This model system will allow identification of the factor(s).

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate.

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.