Genomewide linkage study in the Irish affected sib pair study of alcohol dependence: evidence for a susceptibility region for symptoms of alcohol dependence on chromosome 4.

Alcoholism is a relatively common, chronic, disabling and often treatment-resistant disorder. Evidence from twin and adoption studies indicates a substantial genetic influence, with heritability estimates of 50-60%. We conducted a genome scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD). Most probands were ascertained through alcoholism treatment settings and were severely affected. Probands, affected siblings and parents were evaluated by structured interview. A 4 cM genome scan was conducted using 474 families of which most (96%) were comprised by affected sib pairs. Nonparametric and quantitative linkage analyses were conducted using DSM-IV alcohol dependence (AD) and number of DSM-IV AD symptoms (ADSX). Quantitative results indicate strong linkage for number of AD criteria to a broad region of chromosome 4, ranging from 4q22 to 4q32 (peak multipoint LOD=4.59, P=2.1 x 10(-6), at D4S1611). Follow-up analyses suggest that the linkage may be due to variation in the symptoms of tolerance and out of control drinking. There was evidence of weak linkage (LODs of 1.0-2.0) to several other regions, including 1q44, 13q31, and 22q11 for AD along with 2q37, 9q21, 9q34 and 18p11 for ADSX. The location of the chromosome 4 peak is consistent with results from prior linkage studies and includes the alcohol dehydrogenase gene cluster. The results of this study suggest the importance of genetic variation in chromosome 4 in the etiology and severity of alcoholism in Caucasian populations.

Characterization of DRGs, developmentally regulated GTP-binding proteins, from pea and Arabidopsis.

Developmentally regulated GTP-binding proteins (DRGs) from animals and fungi are highly conserved but have no known function. Here we characterize DRGs from pea (PsDRG) and Arabidopsis (AtDRG). Amino acid sequences of AtDRG and PsDRG were 90% identical to each other and about 65% identical to human DRG. Genomic Southern blotting indicated that AtDRG and PsDRG probably are single-copy genes. PsDRG mRNA accumulated preferentially in growing organs (root apices, growing axillary buds and elongating stems) compared with their non-growing counterparts. At DRG mRNA was relatively abundant in Arabidopsis leaves, stems and siliques, less abundant in flowers and flower buds, and barely detectable in roots. Histone mRNAs are known to accumulate predominantly during S phase of the cell cycle and are markers for proliferating cells. The patterns of histone H2A mRNA accumulation in pea and Arabidopsis organs were very similar to those of DRG mRNAs. An antiserum raised against a PsDRG N-terminal fusion protein recognized 43 and 45 kDa proteins. PsDRG proteins were more abundant in growing pea roots and stems than in non-growing organs, but they were equally abundant in growing and dormant axillary buds. After differential centrifugation, PsDRG proteins were found primarily in the microsomal (150,000 x g pellet) and soluble (150,000 x g supernatant) cell fractions.

Dormancy-associated gene expression in pea axillary buds. Cloning and expression of PsDRM1 and PsDRM2.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots >> root apices; fully-elongated stems > elongating stems), and thus is an excellent "dormancy" marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs.

Effects of oral contraceptives containing different progestogens on sodium-lithium countertransport in normal females.

In view of the lack of unanimity on the effect of long-term intake of combined oral contraceptives (OC) on external sodium-dependent lithium efflux, otherwise known as sodium-lithium countertransport (SLC), we undertook a double-blind study to investigate the possible interaction between SLC and OC in healthy women with regular menstrual cycles. In a group of 17 volunteers, aged 27.0 +/- 1.1 years (mean +/- s.e.m.) and weighing 61.4 +/- 2.0 kg, ingestion of 30 microg ethinyloestradiol + 150 microg desogestrel for 3 months caused an increase in SLC activity from a baseline value of 0.254 +/- 0.017 mmol/lcell x h to 0.274 +/- 0.017 mmol/lcell x h (P = 0.05). The activity of the transport system after 6 months treatment remained higher than at baseline (0.280 +/- 0.016 mmol/lcell x h, P < 0.025) but was comparable to that at 3 months. Blood pressure and weight remained unaltered during the study period. In a comparable group of 16 volunteers of age 26.1 +/- 1.3 years and weight 63.5 +/- 2.1 kg, control SLC activity (0.218 +/- 0.012 mmol/lcell x h) was comparable to that after 3 months (0.215 +/- 0.011 mmol/lcell x h) and 6 months (0.216 +/- 0.011 mmol/lcell x h) intake of 35 microg ethinyloestradiol + 2 mg cyproterone acetate. Body weight and blood pressure remained similarly unchanged. These results not only confirm that long-term intake of OC may cause increased SLC activity, but also suggest that the type of the progesterone in the medication used could be an important determinant of such interaction.

PIP: This double-blind study investigates the possible interaction between sodium-lithium countertransport (SLC) and oral contraceptives (OCs) in healthy women with regular menstrual cycles. 33 healthy females with regular menstrual cycles aged 18-35 years and weighing 43.5-73.0 kg were recruited. Each volunteer was randomly given either a 30 mcg ethinyl estradiol/150 mcg desogestrel combination or a 35 mcg ethinyl estradiol/2 mg cyproterone acetate combination. For each volunteer, blood was taken in the morning of days 15-18 of their menstrual cycle for biochemistry and SLC assay before treatment, and at the same stage of the cycle after 3 and 6 months. The study showed different effects of two different OC preparations in two groups of otherwise comparable volunteers. Administration of ethinyl estradiol/cyproterone acetate combinations for 6 months was not associated with alteration in any of the parameters measured. However, SLC activity increased after 3 and 6 months of ethinyl estradiol/desogestrel medication. These results not only confirm that long-term intake of OCs may cause increased SLC activity, but also suggest that the type of progesterone in the medication used could be an important determinant of such alteration in the activity of the transport system.

The role of thromboxane A2 in increased whole blood platelet aggregation in oral contraceptive users.

Epidemiological studies have shown that oral contraceptives increase the risk of thromboembolic disease in susceptible women however the mechanisms involved are unclear. We investigated whole blood platelet aggregation in 44 women randomly allocated to 6 cycles of treatment with either gestodene (75ug) or desogestrel (150ug) combined with 30ug ethinyloestradiol (EE). The in vitro effects of aspirin and a thromboxane synthetase inhibitor, dazmegrel (UK38485) were also investigated. Oral contraceptive treatment caused a significant increase in collagen, arachidonic acid (AA) and ADP induced whole blood platelet aggregation. PAF induced aggregation was unchanged. There were no significant differences in the levels of platelet aggregation between the desogestrel/30ugEE and gestodene/30ugEE groups. In vitro incubation of platelets with aspirin and dazmegrel prevented the oral contraceptive induced increase in platelet aggregation. Dazmegrel caused an on treatment decrease in PAF induced aggregation in the desogestrel/30ugEE but not the gestodene/30ugEE group. The results of this study indicate that the use of oral contraceptives is associated with an increase in platelet aggregation that is mediated by changes in thromboxane/prostacyclin ratio(TXA2/PGI2). Although no significant differences were found between the two different progestogen combinations, the effects of dazmegrel on PAF induced aggregation suggest a possible difference in the progestogen modifying effects of desogestrel and gestodene which is unmasked when thromboxane synthetase is inhibited.

PIP: In Ireland, researchers randomly assigned 44 healthy women, 18-34 years old, to either the group using the oral contraceptive (OC) containing 30 mcg ethinyl estradiol (EE) and 150 mcg desogestrel (Marviol) or the group using the OC containing 30 mcg EE and 75 mcg gestodene (Femodene) to determine the progestogen's modifying effect on whole blood platelet aggregation. In vitro, they incubated the platelets with aspirin and a thromboxane synthetase inhibitor (dazmegrel) to examine the role of thromboxane (TXA2) in any increased aggregation. The women were recruited from the postnatal clinic of the Coombe Women's Hospital in Dublin. OC use caused a significant increase in collagen-, arachidonic acid- (AA), and ADP-induced whole blood platelet aggregation (p 0.03, 0.001, and 0.01, respectively). It had no effect on PAF-induced aggregation, however. No significant differences in platelet aggregation levels existed between gestodene and desogestrel. Both aspirin and dazmegrel had a significant inhibitory effect on OC-induced increase in platelet aggregation in terms of collagen, AA, and ADP. This suggests that OCs act synergistically with ADP to cause a TXA2 mediated increase in platelet aggregation. Dazmegrel, but not aspirin, caused a decrease in PAF-induced platelet aggregation in the desogestrel/30 mcg EE group only, suggesting a possible difference in the modifying effects of the 2 progestogens, which is revealed when thromboxane synthetase is inhibited. Dazmegrel's inhibitory effect suggests that increased thromboxane synthetase activity plays a role in the OC-induced platelet hyperactivity. Changes in the TXA2/prostacyclin ratio appear to mediate OCs' effect on platelet aggregation.

Mental and physical countermeasures reduce the accuracy of the concealed knowledge test.

The effects of a physical (pressing the toes to the floor) and a mental (counting backward by sevens) countermeasure on the concealed knowledge test (CKT) were examined in a mock crime experiment with 40 subjects. Some knowledgeable subjects were informed about the nature of the CKT and were trained in the use of a countermeasure, whereas others remained uninformed. All subjects were offered a monetary reward if they could produce a truthful outcome. Subjects were tested using standard field techniques and instrumentation. The physical and, to a lesser extent, the mental countermeasures reduced the accuracy of the CKT. These results clearly demonstrate that the CKT has no special immunity to the effects of countermeasures.

Cell cycle regulation during growth-dormancy cycles in pea axillary buds.

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G1 and G2 nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G1, at the G1/S boundary and near the S/G2 boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G1/S boundary enter S within one hour of decapitation. The presence of a cell population arrested in mid-G1 is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G2 and mitosis, cells arrested at G1/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that has been arrested near the S/G2 boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.

Potentiation of a three drug chemotherapy regimen by radiation.

The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.

Endometrial fibrinolytic enzymes in women with normal menstruation and dysfunctional uterine bleeding.

OBJECTIVE: To study fibrinolysis in the endometrium in women with normal menstruation and dysfunctional uterine bleeding (DUB). DESIGN: Tissue plasminogen activator activity (t-PA) and antigen (t-PAAg) and plasminogen activator inhibitor Type 1 antigen (PAI-1) were measured in homogenates of endometrium sampled between 24 and 36 h after the onset of menstruation. SUBJECTS: Women complaining of menorrhagia who had negative findings at clinical examination and curettage had their menstrual blood loss (MBL) measured from the third cycle after D&C. Those with MBL greater than 80 ml per cycle formed the DUB group. MEASUREMENTS: Fibrinolytic enzyme antigen levels were measured with ELISAs. Tissue plasminogen activator activity was assayed by measuring the rate of conversion of Glu-plasminogen to plasmin, using a chromogenic plasmin substrate. CONCLUSIONS: There is a strong positive correlation between endometrial t-PA activity on the second day of menstruation and measured menstrual loss (P < 0.05). Concentrations of endometrial t-PAAg and PAI-1 antigen are higher in women with DUB compared with normal women during menstruation.

Menstrual blood loss measurement with gynaeseal.

The diagnosis of menorrhagia is usually based on the subjective complaint of heavy menstrual bleeding, although up to 50% of women describing menorrhagia have measured menstrual loss within normal limits. Treatment is usually started without first establishing an objective diagnosis, because menstrual blood loss measurement is not widely available to clinicians. Current laboratory methods of measuring menstrual loss involve extraction of menses from sanitary wear. Many women find collection of sanitary wear unacceptable and laboratory staff find the menstrual extraction procedure unpleasant and time-consuming. We investigated the use of Gynaeseal, a vaginally placed latex menstrual seal, in women with normal menstrual loss (n = 10) and menorrhagia (n = 12) with regard to its suitability for the measurement of menstrual loss and efficacy as alternative sanitary protection. Twenty-one of the 22 women found the device easy to insert, but 16 found it messy to remove. All of the 6 couples having coitus found the device caused no discomfort. All women with menorrhagia and 4 of 12 women with normal menstrual losses were dissatisfied with the menstrual seal provided by gynaeseal. Gynaeseal does not contain menstrual blood efficiently in women with menorrhagia and is therefore unsuitable for the measurement of menstrual blood loss.

Quantitative changes in tumor metabolism, partial pressure of oxygen, and radiobiological oxygenation status postradiation.

Hypoxia is considered to be a major cause of tumor radioresistance. Reoxygenation of previously hypoxic areas after a priming dose of radiation is associated with an increase in tumor radiosensitivity. In a study of a hypoxic mammary carcinoma, 31P nuclear magnetic resonance spectra showed statistically significant increases in metabolite ratios (phosphocreatine/Pi and nucleotide triphosphate/Pi) after 65 and 32 Gy. The maximum changes in metabolite ratios after 32 Gy occurred at 48 h, although significant changes were detected at 24 h. A corresponding increase in the mean tumor pO2 (polarographic microelectrode measurements) and a decrease in hypoxic cell fraction [changes in paired (clamped versus unclamped) tumor control dose for 50% of tumors] were also shown to occur 48 h after a priming dose of 32 Gy. A significant increase in the mean tumor pO2, phosphocreatine/Pi, and nucleotide triphosphate/Pi, compared to initial values, was noted at 24, 48, and 96 h post 65-Gy radiation. An increase in the downfield component of the phosphomonoester peak relative to the upfield component (phosphoethanolamine), is also noted after doses of 65 and 32 Gy. These are likely to be due to cell kill and/or decreased cell proliferation. In this tumor model, 31P nuclear magnetic resonance spectroscopic changes postradiation are temporally coincident with and may be indicative of tumor reoxygenation as measured by the tumor control dose for 50% of tumors and oxygen-sensitive microelectrodes.

Indomethacin and ethamsylate alone and in combination for the relief of post episiotomy pain.

In a bid to minimise dosage and possible side-effects when relieving post episiotomy pain, the NSAID Indomethacin was studied in combination with a systemic haemostat Ethamsylate which has been shown to selectively inhibit some prostaglandins. Comparative groups also took Indomethacin alone and placebo in a double blind non-crossover comparison. Efficacy was judged in terms of side-effects and assessments of pain intensity, pain relief and global assessment of pain. There was some evidence of a beneficial interaction between Indomethacin and Ethamsylate when adjustments were made for the patient's age and initial pain score. Side-effects were most common in the combination therapy group.

Quantitation of total metastatic tumor volume in the rat liver: correlation of MR and histologic measurements.

Assessing tumor response to chemotherapy in the liver has always been difficult. Most investigators estimate tumor volume as either a product of the two perpendicular diameters of a tumor nodule, or, in animal studies, simply count surface tumor nodules. The authors evaluated magnetic resonance (MR) imaging as a technique for determining absolute tumor volume in the liver in an animal model. Specifically, histologic volumetric and MR imaging measurements of tumor and liver volumes were quantitatively compared over a wide range of tumor burdens in a rat model of hepatic metastasis of a colorectal carcinoma. Twenty-three rats were imaged, with two different section thicknesses used in each animal. Both section thicknesses showed highly significant correlations between MR and histologic measurements for both tumor and liver volumes (P less than .001). MR imaging may be useful for noninvasively quantifying tumor burden and temporal response of metastatic disease in the liver to novel antineoplastic regimens.

Health record completion guidelines. American Health Information Management Association.

It is a pleasure to introduce this important project report to the American Health Information Management Association (AHIMA) membership. Analyzing records for omissions, notifying physicians of needed information, counting delinquent records, and pursuing late documentation are some of the biggest chores in today's health information management departments. And they are chores that take time away from other priorities--managing, analyzing, and presenting health data, planning and implementing computerization, assessing and meeting customer needs. The heart of this statement is simple: it points out that there are other options to the traditional, detailed, record-by-record analysis. And those options may give us the results we need--timely and complete health records--while freeing up valuable staff time for other priorities. Take a serious look at the statement. If you are eager to make a change in your department's practices in records analysis and completion, it will back you up. If you are comparing the value of your department's records completion work to its benefit, this statement will give you ideas for change. And if you don't think you'd ever challenge tradition, this statement will give you food for thought. An added value to this statement is the fact that the ideas in it, and the very statement itself, are the product of our own profession. We are fortunate that leading-edge practitioners gave their expertise to the entire profession. The members of the strategy group for this project are listed above, we thank them for their wisdom.

In vivo monitoring of changes in 5-fluorouracil metabolism induced by methotrexate measured by 19F NMR spectroscopy.

We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.

Changes in radiation sensitization induced by Fluosol-DA as measured by 31P nuclear magnetic resonance spectroscopy.

Numerous agents have been studied in attempts to sensitize radioresistant hypoxic tumor cells. We have investigated the effect of Fluosol-DA plus carbogen (95% oxygen and 5% CO2) on the sensitivity of a radioresistant mammary carcinoma in C3H/He mice and also on tumor metabolism by 31P nuclear magnetic resonance spectroscopy. Statistically significant increases in phosphocreatine/Pi were noted for small- (150-350 mm3) and medium- (351-650 mm3) sized tumors treated with Fluosol-DA plus carbogen. Small tumors were shown to undergo significant radiosensitization in the presence of Fluosol-DA plus carbogen and medium-sized tumors showed a lesser degree of radiosensitization. Large tumors (greater than 900 mm3) showed no effect. Fluosol-DA or carbogen alone had no effects on animals with any tumor volume, as monitored by significant changes in radiosensitivity or nuclear magnetic resonance parameters. An approximately linear relationship was found between the decrease in the values for radiation dose which yields 50% tumor control and the increase in phosphocreatine/Pi, with a correlation of r = -0.93. 31P nuclear magnetic resonance spectroscopy may be useful for monitoring changes in radiosensitivity induced by agents which alter tumor oxygenation and subsequent metabolic status.

Fatty acyl composition of the major lipid classes of HDL2 and HDL3 of human serum.

High density lipoprotein subfractions 2 and 3 were isolated from the sera of normolipidaemic males by rate zonal ultracentrifugation. The fatty acyl compositions of their major lipid classes--phospholipids, cholesteryl esters and triacylglycerols--were determined. The average chain length and degree of unsaturation of the fatty acyl components of the phospholipids and cholesteryl esters of the HDL3 subfraction were found to be significantly higher than those of the HDL2 subfraction. The triacylglycerol moieties of the HDL3 subfraction had a higher proportion of unsaturated fatty acyl components than HDL2, but there was no difference in the average chain length between the two subfractions. These results are not consistent with an equilibration of the lipid components between the two HDL subfractions, as would be expected from the results of tracer studies. A role for the fatty acyl composition of high density lipoproteins in the determination of high density lipoprotein subfraction distribution is proposed.