Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort.

BACKGROUND: Inherited thrombophilias have inconsistently been linked to adverse pregnancy outcomes. Differences in study design, size and population could explain this heterogeneity. OBJECTIVE: The aim of the present study was to evaluate if factor (F)V Leiden G1691A, prothrombin mutation G20210A (PTM) and methylenetetrahydrofolate reductase C677T (MTHFR) increased the risk of severe preeclampsia, fetal growth restriction, very preterm delivery, placental abruption and a composite of these outcomes also including stillbirth. PATIENTS AND METHODS: In a nested case-cohort study of pregnant women in Denmark, we genotyped 2032 cases and 1851 random controls. Each of the medical records of the cases was validated. We calculated both genomic and allelic models, and present both models. We also performed sensitivity analyses adjusting for parity, age, smoking, body mass index and socioeconomic status. RESULTS: In the allelic models, FV Leiden increased the risk of the composite outcome (odds ratio [OR] 1.4, 95% confidence interval [CI] 1.1-1.8), severe preeclampsia (OR 1.6, 95% CI 1.1-2.4), fetal growth restriction (OR 1.4, 95% CI 1.1-1.8) and placental abruption (OR = 1.7 (95% CI 1.2-2.4). In the sensitivity analyses, adjustment diminished these estimates slightly. PTM was not significantly associated with any of the outcomes, and MTHFR was only significantly associated with severe preeclampsia (OR 1.3, 95% CI 1.1-1.6). CONCLUSION: FV Leiden predisposes to adverse pregnancy outcomes in a setting of Scandinavian women.

Genetic variants associated with deep vein thrombosis: the F11 locus.

BACKGROUND: Recent studies have found associations between deep vein thrombosis (DVT) and single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that contains genes encoding factor XI (F11), a cytochrome P450 family member (CYP4V2), and prekallikrein (KLKB1). OBJECTIVE: We investigated which of the common SNPs in this locus are independently associated with DVT. METHODS: The study populations were the Leiden Thrombophilia Study (LETS) (443 DVT cases and 453 controls) and the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study) (2712 DVT cases and 4634 controls). We assessed the association between DVT and 103 SNPs in a 200 kb region using logistic regression. RESULTS: We found that two SNPs (rs2289252 and rs2036914 in F11) were independently associated with DVT. After adjusting for age, sex, and the other SNP, the odds ratios (risk vs. non-risk homozygotes) of these two SNPs were 1.49 for rs2289252 (95% CI, 1.25-1.76) and 1.33 for rs2036914 (95% CI, 1.11-1.59). We found that rs2289252 was also associated with FXI levels, as has been previously reported for rs2036914; these two SNPs remained associated with DVT with somewhat attenuated risk estimates after adjustment for FXI levels. CONCLUSION: Two SNPs, rs2289252 and rs2036914 in F11, appear to independently contribute to the risk of DVT, a contribution that is explained at least in part by an association with FXI levels.

Proof of principle of potential clinical utility of multiple SNP analysis for prediction of recurrent venous thrombosis.

BACKGROUND: Individual single nucleotide polymorphisms (SNPs) associated with an increased risk of a first venous thrombosis do not predict risk of recurrent thrombosis. OBJECTIVE: To assess the risk of recurrent venous thrombosis associated with multiple SNPs. PATIENTS/METHODS: Fifteen nucleotide polymorphisms (SNPs), either established or putative risk factors for venous thrombosis, were measured in 817 unselected patients presenting with a first episode of venous thrombosis. Data from patients enrolled in the Leiden Thrombophilia Study (LETS) (n = 443) and the first Cambridge Prospective Cohort Study (n = 374) were combined. Hazard ratios for recurrence of thrombosis were calculated for individual SNPs. RESULTS: Of the total study population, 117 patients had a recurrent event after a mean follow-up of 4.6 years. The overall incidence rate was 30.8/1000 person years, corresponding with an annual risk of 3.1%. None of the individual SNPs was more than weakly associated with the risk of recurrent venous thrombosis. With addition of sequential SNPs, added in rank order of risk, the hazard ratios for recurrence increased by 1.7-fold for carriers (3.8% of all patients) of the first two SNPs, 2.7-fold for carriers of three (2.3%) and 5.1-fold for carriers of four (0.4%). With addition of each SNP the number of carriers rapidly reduced. CONCLUSIONS: Although there is a substantially increased risk of recurrent thrombosis for carriers of several genetic variants, the clinical utility of multiple SNP analysis at present would be limited to a small proportion of patients.

Use of enhanced green fluorescent protein to optimize and quantitate infection of target cells with recombinant retroviruses.

Recombinant retroviral vectors are useful tools for gene transfer in both gene therapy and research applications. An enhanced form of green fluorescent protein has been incorporated into recombinant retroviruses as a marker to follow infected cells. In this paper, we extended the use of the fluorescent reporter to quantify protein expression using such analytical tools as fluorescent microscopy, flow cytometry and fluorescent plate reader analysis. These tools enabled us to rapidly assess the titer of recombinant retrovirus harvested from packaging cells and to optimize parameters for infection of different cell lines.

Merkel cell carcinoma: the use of surgery and radiotherapy for locoregional control.

Merkel cell tumours are rare skin cancers with an unpredictable clinical course. Correct diagnosis requiring immunohistochemical staining is essential since the treatment differs entirely from that of basalioma. Locally limited disease as well as rapidly fatal course have been observed. In a small series of 5 patients and from literature data, surgery continued with radiotherapy for locoregional control is advocated as optimal approach since this type of tumour can behave differently compared to basal cell carcinomas.

Identification of motilin mRNA in the brain of man and rabbit. Conservation of polymorphism of the motilin gene across species.

The data regarding the identity of motilin-like immunoreactivity in the central nervous system are controversial. The aim of the present study was to clarify whether motilin mRNA is present in the brain of rabbit and man. Total RNA, prepared from several regions of the rabbit brain, was subjected to RT-PCR aimed at amplifying a 294 bp cDNA fragment of the rabbit motilin precursor. The amplified product was subcloned and sequenced. The sequence showed 7 differences compared to the one reported for the duodenal precursor (1). However the duodenal precursor from the rabbit used in the present study revealed identical substitutions. One of these, involving amino acid -11 of the signal peptide, was shown to be due to gene polymorphism, as has also been described at this site in man. By radioimmunoassay the highest concentration of motilin (fmol/mg protein) was detected in the hippocampus (4788 +/- 295), the lowest in the telencephalon (2127 +/- 221). Using a similar approach, but starting from commercial human brain mRNA, the sequence of a comparable cDNA fragment of the human brain motilin precursor was obtained. Its sequence was identical with the one published for the human intestinal precursor (41). Our study demonstrates that motilin mRNA is present in the brain of man and rabbit. Together with our recent findings of central motilin receptors, they suggest a central role for motilin.

Effects of resistance training on regional and total bone mineral density in premenopausal women: a randomized prospective study.

This study was designed to assess the effects of 18 months of resistance exercise on regional and total bone mineral density (BMD) and soft tissue lean mass (STL) in premenopausal women aged 28-39 randomly assigned to an exercise or control group. Twenty-two exercise and 34 control subjects completed the 18-month training study. All subjects were previously inactive and untrained women. Initial, 5-, 12- and 18-month assessments were made of total and regional BMD and total and regional STL using dual energy X-ray absorptiometry. All subjects consumed a 500 mg/day elemental calcium supplement throughout the study. Initial Ca intake without supplement averaged 1,023 mg/day in total sample. Serum levels of bone osteocalcin and dietary assessments using 12 randomly assigned days of diet records were also completed. Muscular strength was assessed from both 1 repetition maximum (RM) testing of 10 weightlifting exercises and by peak torque for hip abduction/adduction and knee extension/flexion. Training increased strength by 58.1% based on 1 RM testing and by 33.8% based on isokinetic testing at 18 months versus baseline. BMD increased significantly above baseline at the lumbar spine for the exercise group at 5 months (2.8%), 12 months (2.3%), and 18 months (1.9%) as compared with controls. Femur trochanter BMD increased significantly (p < 0.05) in the exercise group at 12 months (1.8%) and 18 months (2.0%) but not at 5 months (0.7%) as compared with controls. No changes in total BMD, arm BMD, or leg BMD were found. There was a 20% increase in BGP in the exercise group as compared with controls at 5 months and this difference was maintained throughout the study. For STL, significant increases for total, arm, and leg were found at 5, 12, and 18 months for the exercise group versus control ranging from 1-6% over baseline. These results support the use of strength training for increasing STL and muscular strength with smaller but significant regional increases in BMD in the premenopausal population.

Effect of cysteine substitutions on the mitogenic activity and stability of recombinant human keratinocyte growth factor.

Human Keratinocyte growth factor (hKGF), a member of the FGF family of growth factors, contains five cysteines at amino acid positions 1, 15, 40, 102, and 106. We expressed five cysteine mutants of hKGF in which the cysteines were cumulatively replaced with alanine or serine, starting with cysteine-1. Recombinant hKGF has an inherently higher mitogenic activity and stability to heat and acid than reported for glycosylated hKGF. Mitogenic activity is increased an additional 2.6 fold by substitution of cysteine-1 with alanine. Mutants with the conserved cysteine substituted at position 40 were more susceptible to heat inactivation than rhKGF, but showed no significant difference in acid inactivation. Cysteine-free rhKGF is mitogenic, demonstrating that neither cysteines nor disulfide bonds are required for mitogenic activity. However, cysteine-free rhKGF does not bind Heparin-Sepharose and is unstable to heat and acid compared to rhKGF, suggesting that the cysteines have a role in maintaining KGF's structure. This information will useful in the development of a more stable and more potent wound healing agent from hKGF.

Expression of two variants of the human mu opioid receptor mRNA in SK-N-SH cells and human brain.

A partial mu opioid receptor gene was isolated from a human genomic library using a mouse delta opioid receptor cDNA as a probe. Using information from this genomic clone and the published human mu receptor, MOR1, a cDNA was isolated from SK-N-SH mRNA that codes for a variant of the MOR1 mRNA, MOR1A. The presence of MOR1A is also shown in human brain using RT-PCR. MOR1A differs from MOR1 in that the 3' terminal intron has not been removed. An in-frame termination codon is found four amino acids after the 5' consensus splice site, making MOR1A eight amino acids shorter than MOR1. Both receptors show similar ligand binding and coupling to cAMP in CHO-K1 cells. The C-terminal differences between MOR1 and MOR1A could have effects on receptor coupling or receptor transport and localization.

Relationship between severity, costs and claims of hospitalized patients using the Severity of Illness Index.

The results of the prospective application of Horn's 'Severity of Illness Index' in a teaching hospital during 1987, 1989, and 1990 constitute the basis of the present report. The average overall severity of illness scores for the three years were 1.42 in 1987, 1.65 in 1989, and 1.46 in 1990. Most of the processes evaluated in the three periods showed an overall distribution among severity levels 1 and 2, both overall and when the seven dimensions of the severity of illness index were analyzed. A statistically significant correlation between the overall severity of illness and average length of stay was found for patients in 1989 and 1990. The length of stay differed significantly in the different severity levels. When the four levels of the seven dimensions of the severity of illness index for 1987, 1989, and 1990 were compared, it was observed that figures were not uniformly distributed. There was a statistically significant association between severity of illness for hospital service and pharmacy charges per hospital stay for both 1989 and 1990, as well as a statistically significant inverse relationship between severity of illness and the number of claims per hospital service in both periods of time. Case-mix methods that account for the severity of patients constitute a useful indicator of quality for the management of different hospital services and of the hospital as a whole.

Isolation of a human kappa opioid receptor cDNA from placenta.

By using a mouse delta opioid receptor cDNA as a probe, a kappa opioid receptor gene was isolated from a human genomic library. Reverse transcriptase/PCR was subsequently used to isolate the corresponding cDNA from human placenta mRNA. Characterization of the cloned receptor with a kappa agonist in transfected COS-7 cells, revealed a Kd of 0.67 nM, which is similar to rodent kappa 1 receptors. In competition binding experiments, non-kappa agonists had kis > 1000nM. Furthermore, when expressed in COS-7 cells, the kappa receptor is negatively linked to cAMP. A Northern blot showed the presence of two transcripts, 6 and 7 kB in size, in both placenta and brain mRNA, which hybridized to the kappa cDNA.

Prediction of body composition in premenopausal females from dual-energy X-ray absorptiometry.

Four methods for predicting body composition were compared in premenopausal females (n = 100), 28-39 yr old, by using underwater weighing (UWW) as the criterion method. The four methods were dual energy X-ray absorptiometry (DEXA), skinfolds, bioelectrical impedance, and body mass index. The sample had a mean percent fat (%fat) of 29.7 +/- 6.8% (SD) by DEXA and 29.9 +/- 5.8% measured by UWW. DEXA yielded a standard error of estimate (SE) of 2.4% (r = 0.91) for the prediction of %fat from UWW. When %fat was estimated from other methods, larger SEs were obtained: 3.0% for skin-folds, 3.3% for body mass index, and 2.9% for bioelectrical impedance (height2/resistance) plus weight. Individual body density values derived from UWW were corrected for bone mineral variation. DEXA predicted the corrected body density with a lower SE (0.0040 vs. 0.0053 g/ml) than the original density values. We conclude that DEXA was a precise method and correlated highly with fat-free body weight and %fat from UWW in this homogeneous female sample.

Detection of small changes in body composition by dual-energy x-ray absorptiometry.

The ability of dual-energy x-ray absorptiometry (DEXA) to detect small changes in body composition was studied in 17 men and women during a dehydration-rehydration protocol. Scale weight (BW) and total mass (TM) from DEXA were highly related (r > 0.99) as were estimates of fat-free mass (r = 0.99) and percent fat (r = 0.97) from DEXA and densitometry. Changes in BW of approximately 1.5 kg due to fluid loss and gain were highly correlated (r = 0.90) with both changes in TM and soft-tissue mass (STM) by DEXA but less so (r = 0.67) with changes in lean-tissue mass (LTM). Mean changes in TM, STM, and LTM were not different (P > 0.05) from changes in BW. Estimates of bone mass and fat were unaffected by changes in hydration. We conclude that DEXA is able to detect small individual changes in TM and STM and is also useful for detecting group changes in LTM.

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

Specific substitution into the anticodon loop of yeast tyrosine transfer RNA.

The aminoacylation kinetics of 19 different variants of yeast tRNATyr with nucleotide substitutions in positions 33-35 were determined. Substitution of the conserved uridine-33 does not alter the rate of aminoacylation. However, substitution of the anticodon position 34 or position 35 reduces Km from 2- to 10-fold and Vmax as much as 2-fold, depending on the nucleotide inserted. The ochre and amber suppressor tRNAsTyr both showed about a 7-fold reduction in Vmax/Km. Data from tRNATyr with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of psi-35. The effect of simultaneous substitutions at positions 34 and 35 can be predicted reasonably well by combining the effects of single substitutions. These data suggest that yeast tyrosyl-tRNA synthetase interacts with positions 34 and 35 of the anticodon of tRNATyr and opens the possibility that nonsense suppressor efficiency may be mediated by the level of aminoacylation.

Aminoacylation of anticodon loop substituted yeast tyrosine transfer RNA.

A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.

Uridine-33 in yeast tRNA not essential for amber suppression.

The nucleotide at position 33 on the 5' side of the anticodon of almost all tRNAs is a uridine. Crystallographic studies of different tRNAs reveal that although the precise orientation of uridine-33 is not always the same, it connects the anticodon stacked along the 3' side of the loop with the pyrimidine-32 stacked on the 5' side of the loop. The remarkably conserved nature of uridine-33 and its unique position in the anticodon loop structure has led to suggestions that this nucleotide has an essential role in the translational mechanism. We have developed a biochemical procedure to replace nucleotides 33-35 in yeast tRNATyr with any desired sequence and used it to construct amber suppressor tRNAs having different nucleotides at position 33. As all of these synthetic amber suppressor tRNAs functioned well in eukaryotic in vitro suppression assays, we conclude that uridine-33 does not have an obligatory role in the translation mechanism.