Observational follow-up of the PROactive study: a 6-year update.

AIMS: The PROactive study investigated pioglitazone for secondary prevention of macrovascular events in type 2 diabetes mellitus. Pioglitazone showed a 10% (non-significant) relative risk (RR) reduction for the primary composite endpoint and a significant 16% reduction for the main secondary endpoint (death, myocardial infarction, stroke) after a mean 34.5 months. There was no difference in cumulative malignancy incidence, but an imbalance in bladder malignancies (pioglitazone 14, placebo 5). We present a pre-specified 6-year interim analysis of a 10-year observational follow-up. METHODS: Any patient completing PROactive was eligible. No study treatments were provided. A Cox proportional hazard model compared non-adjudicated macrovascular events (same endpoints as PROactive excluding acute coronary syndrome) based on original randomization. Malignancies were compared using conventional RR ratios. RESULTS: Of 5238 randomized patients, 3599 (74%) entered the follow-up. For the follow-up (mean 5.8 years) or combined double-blind and follow-up periods (≤9.5 years, mean 8.7), there were no statistically significant differences in primary or main secondary endpoints. For the combined period, a similar percentage of patients had any diagnosed malignancy (RR = 1.05, 95% CI [0.89, 1.24]) or bladder malignancy (RR = 1.06, 95% CI [0.59, 1.89]) in the pioglitazone and placebo groups. There were fewer cases of bladder malignancy with pioglitazone (15 [0.6%] vs. 19 [0.7%] for placebo) for the combined period when events diagnosed in the first 365 days were excluded, and fewer cases for the follow-up period alone (10 [0.5%] vs. 17 [1.0%] for placebo). Further analyses of pioglitazone use (including use during follow-up) found no significant difference in bladder malignancies between any and no pioglitazone use for the combined period. CONCLUSIONS: These data suggest that improved macrovascular outcomes seen with pioglitazone subside without continued pioglitazone treatment. The double-blind period bladder cancer imbalance did not persist in follow-up.

Detection and imaging of atmospheric radio flashes from cosmic ray air showers.

The nature of ultrahigh-energy cosmic rays (UHECRs) at energies >10(20) eV remains a mystery. They are likely to be of extragalactic origin, but should be absorbed within approximately 50 Mpc through interactions with the cosmic microwave background. As there are no sufficiently powerful accelerators within this distance from the Galaxy, explanations for UHECRs range from unusual astrophysical sources to exotic string physics. Also unclear is whether UHECRs consist of protons, heavy nuclei, neutrinos or gamma-rays. To resolve these questions, larger detectors with higher duty cycles and which combine multiple detection techniques are needed. Radio emission from UHECRs, on the other hand, is unaffected by attenuation, has a high duty cycle, gives calorimetric measurements and provides high directional accuracy. Here we report the detection of radio flashes from cosmic-ray air showers using low-cost digital radio receivers. We show that the radiation can be understood in terms of the geosynchrotron effect. Our results show that it should be possible to determine the nature and composition of UHECRs with combined radio and particle detectors, and to detect the ultrahigh-energy neutrinos expected from flavour mixing.

Improved flux limits for neutrinos with energies above 10(22) eV from observations with the Westerbork Synthesis Radio Telescope.

Particle cascades initiated by ultrahigh energy neutrinos in the lunar regolith will emit an electromagnetic pulse with a time duration of the order of nanoseconds through a process known as the Askaryan effect. It has been shown that in an observing window around 150 MHz there is a maximum chance for detecting this radiation with radio telescopes commonly used in astronomy. In 50 h of observation time with the Westerbork Synthesis Radio Telescope array we have set a new limit on the flux of neutrinos, summed over all flavors, with energies in excess of 4x10(22) eV.

[Diagnostic image (332). A young woman with amenorrhoea]. / Diagnose in beeld (332). Een jonge vrouw met amenorroe.

A 16-year-old girl presented with amenorrhoea and a tense abdomen, due to a mature cystic teratoma with the characteristics ofa dermoid cyst.

Cardiovascular determinants of carotid artery disease. The Rotterdam Elderly Study.

The objective of the present study was to assess the prevalence of moderate and severe stenosis of the right carotid artery in the elderly and its associations with smoking, blood pressure, serum lipid levels, and hemostatic factors. The Rotterdam Elderly Study is a recently started single-center prospective follow-up study of a cohort of 11,854 elderly people aged 55 years or more. In 1990, 954 participants of the Rotterdam Elderly Study underwent ultrasonic duplex examination of the right internal carotid artery. A reduction of the lumen diameter of 16-49% was found in 29 people (3.0%). Severe stenosis (50% or more) was observed in 13 people (1.4%). With differences in age, sex, and body mass index taken into account, subjects with moderate-to-severe carotid artery disease had, compared with participants without stenosis, lower mean high density lipoprotein cholesterol levels (mean difference, 0.10 mmol/l; 95% confidence interval, 0, 0.20) and higher mean fibrinogen levels (difference, 0.24 g/l; 0.04, 0.45). Among them were more people with hypertension (mean difference, 16%) and more current smokers (mean difference, 13%). Factor VIIc and factor VIIIc activity was higher in subjects with carotid artery disease, without, however, reaching statistical significance (mean difference, 0.06 IU/ml [-0.01, 0.12] and 0.21 IU/ml [-0.05, 0.47], respectively). Our data suggest that hypertension, smoking, and reduced serum high density lipoprotein cholesterol levels, combined with unfavorable increases in hemostatic factors, may be related to carotid artery disease in the elderly.

Linkage analysis of families with bipolar illness and chromosome 18 markers.

Linkage of bipolar (BP) illness with chromosome 18 markers located at 18p11 was recently reported. A possible role for chromosome 18 in the etiology of BP illness was implicated previously by the finding in three unrelated patients of a ring chromosome with breakpoints and deleted segments at 18pter-p11 and 18q23-qter. To test the potential importance of a gene defect on chromosome 18 in our material, we examined linkage with chromosome 18 markers in two families with multiple patients with BP illness or BP spectrum disorders. fourteen simple tandem repeat polymorphisms were used located in the chromosomal region 18p11 to 18q23 and separated by distances of approximately 10 cM on the genetic map. In one family linkage to chromosome 18 could not be excluded. Linkage and segregation analysis in the family suggests that the 12-cM region between D18S51 and D18S61 located at 18q21.33-q23 may contain a candidate gene for BP illness.

Expanded trinucleotide CAG repeats in families with bipolar affective disorder.

Clinical anticipation has been reported in bipolar affective disorder (BPAD). The hypothesis that expanded trinucleotide repeats are related to anticipation and transmission pattern in families with bipolar affective disorder is tested in this study. Eighty-seven two-generation pairs of patients recruited from 29 bipolar families were analyzed. The repeat expansion detection method was used to detect CAG repeat expansions between successive generations. Significant changes in age at onset and episode frequency in successive generations were observed. Mean trinucleotide CAG repeat length between parental and offspring generation significantly increased when the phenotype increased in severity, i.e., changed from major depression, single episode or unipolar recurrent depression to BPAD. A parent-of-origin effect was also observed with a significant increase in median length CAG between G1 and G2 with maternal inheritance. This increase was observed notably in female offspring. Our findings indicate for the first time that expansion of CAG repeat length could explain the clinical observation of anticipation in families with BPAD. These results provide further support for expanded trinucleotide repeat sequences as risk factors in major affective disorders.

Absence of linkage between chromosome 11p15 markers and manic-depressive illness in a Belgian pedigree.

OBJECTIVE: The original finding of genetic linkage in an Old Order Amish pedigree has been contradicted by the results of several subsequent studies. Using the same genetic parameter values, diagnostic criteria, and 11p15 genetic markers as those used to study the initial Amish population, the authors performed a linkage study of a four-generation informative pedigree in Belgium. METHOD: Recombinant DNA technology was used to analyze three markers for the chromosome 11p15 location: the genes for tyrosine hydroxylase (TH) and insulin (INS) and the c-Harvey-ras oncogene (HRAS). Diagnoses of the relatives of a proband with bipolar affective disorder were determined with the Schedule for Affective Disorders and Schizophrenia--Lifetime Version and based on the Research Diagnostic Criteria. Relatives were considered affected if they had bipolar disorder, unipolar disorder, or cyclothymia; a diagnostic hierarchy was developed to include unipolar disorder and cyclothymia in the linkage analysis. RESULTS: Pairwise analyses of the disease locus and each of the three polymorphisms excluded the possibility of close linkage between manic-depressive illness and the three chromosome 11p15 markers. Multipoint linkage analysis combining the information from all three genes also excluded linkage. CONCLUSIONS: The conflict between the original results from the Amish study and the many negative reports on chromosome 11 linkage of manic-depression has been interpreted as indicating genetic heterogeneity, but heterogeneity has not been documented for the 11p15 locus. Conversely, the linkage approach has major drawbacks, so other genetic strategies should also be considered.

Nonlinkage of bipolar illness to tyrosine hydroxylase, tyrosinase, and D2 and D4 dopamine receptor genes on chromosome 11.

OBJECTIVE: Previous linkage and allelic association studies using DNA polymorphisms, cosegregation of cytogenetic abnormalities with psychiatric illness, and assignment of genes involved in neutotransmitter metabolism suggested that chromosome 11 may harbor a gene predisposing to bipolar illness. The authors examined linkage in the families of 14 probands with bipolar illness, with the candidate genes tyrosine hydroxylase (TH), D4 dopamine receptor (DRD4) at 11p15, tyrosinase (TYR) at 11q14-q21, and D2 dopamine receptor (DRD2) at 11q22-q23, as well as with the c-Harvey-ras oncogene (HRAS) and insulin gene (INS), both located at 11p15, a region that previously showed linkage to bipolar illness. METHOD: The genetic data were analyzed with both lod score analysis (parametric) and affected-sib-pair analysis (nonparametric); both narrow and broad definitions of the clinical phenotype were used. Further influences of diagnostic uncertainties were accounted for by using diagnostic probability classes weighing the stability of each phenotype. RESULTS: Two-point linkage results excluded close linkage of bipolar illness to each candidate gene; negative results were also obtained when the narrow definition of the clinical phenotype was used. Moreover, multipoint linkage analysis of HRAS and INS excluded the 11p15 region encompassing both DRD4 and TH. In agreement with the negative linkage results, affected-sib-pair analysis did not show preferential sharing of marker alleles at any of the candidate genes. CONCLUSIONS: The negative results obtained under different genetic models exclude a frequent role for DRD4, TH, TYR, and DRD2 in the pathogenesis of bipolar illness.

NMR evidence for a novel asparagine-linked oligosaccharide on cellobiohydrolase I from Trichoderma reesei RUTC 30.

The primary structure of a novel phosphate-containing oligosaccharide, isolated from T. reesei cellobiohydrolase I, was determined by NMR techniques. The new compound has the same structure as GlcMan7GlcNAc2, but it is extended by one alpha-mannopyranosyl unit (Man-P) through a phosphate link. Three different heteronuclear (31P-1H) NMR techniques were used to prove that the phosphate links the glycosidic site of Man-P with C-6 of unit Man-B. The presence of mannoses linked through a phosphate diester resembles glycosyl synthesis in yeast.

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei.

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

The validity of the radial immunodiffusion method for the quantitative determination of human IgM. Evaluation of a modified method.

Three subgroups of human IgM can be distinguished on the basis of differences of slopes of the D2 versus absolute concentration plot in the radial immunodiffusion technique (Klein et al., 1973). Individual IgM fractions, whether mono- or polyclonal, always belong to only one of these groups. The differences between the subgroups disappear after reduction of the IgM to 7S subunits. These findings provide an explanation for the large discrepancies between absolute IgM determinations in different laboratories. It follows that most of the readings of individual IgM values in the Mancini technique must be wrong by any standard, including WHO reference preparations. The insertion of a simple reduction step in the assay abolishes the differences in quantitative reactivity between IgM subgroups as well as between natural 7S and 19S IgM. This allows an absolute determination of both forms together. The values thus obtained differ considerably from the estimates given by Humphrey and Batty (1974). It also appears that the International Units of the WHO do not represent the same quantity of IgM in different reference sera. The modified method allows the determination of total monoclonal as well as polyclonal human IgM by weight.

The absolute quantification of human IgM and IgG: standardization and normal values.

Nephelometric, turbidimetric and radial immunodiffusion methods are described, which permit valid, reproducible determination of human IgM and IgG by weight. Subgroups of IgM, 7S-IgM and subclasses of IgG did not interfere with these methods. For IgG the International Unit represented a constant weight quantity in agreement with official WHO estimations. For IgM the IU was not constant and much lower values are found. It is therefore suggested that the IU should be abolished for human IgM and IgG. Absolute values for IgM levels are presented for both sexes and for age groups in the range of 5-80 years. The figures showed significant variation with sex and age.

Synthesis and structure-activity relationships of analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a selective inhibitor of trypanosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase.

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction of a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre.

N6-cyclopentyl-3'-substituted-xylofuranosyladenosines: a new class of non-xanthine adenosine A1 receptor antagonists.

The present study explores the C-3' site of the 3-deoxy-3-xylofuranosyl ring of nucleoside analogues with an adenine or N6-cyclopentyladenine (CPA) base moiety and evaluates the effect on adenosine receptor affinity. Two series of sugar-modified adenosines, i.e., 3'-amido-3'-deoxyadenosines and 3'-amidated 3'-deoxyxylofuranosyladenines, were synthesized and tested for their affinity at A1 and A2a receptors in rat brain cortex and rat striatum, respectively. The modest affinity found in the "xylo series" prompted us to synthesize the corresponding N6-cyclopentyl derivatives, which proved to be well accommodated by the A1 receptors with potencies in the lower nanomolar range. This represents a new perspective in the purinergic field. The absence of a GTP-induced shift, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP indicates an antagonistic behavior of this new class of CPA analogues.

Linkage analysis of bipolar illness with X-chromosome DNA markers: a susceptibility gene in Xq27-q28 cannot be excluded.

Transmission studies have supported the presence of a susceptibility gene for bipolar (BP) illness on the X-chromosome. Initial linkage studies with color blindness (CB), glucose-6-phosphate dehydrogenase (G6PD) deficiency, and the blood coagulation factor IX (F9) have suggested that a gene for BP illness is located in the Xq27-q28 region. We tested linkage with several DNA markers located in Xq27-q28 in 2 families, MAD3 and MAD4, that previously were linked to F9 and 7 newly ascertained families of BP probands. Linkage was also examined with the gene encoding the alpha 3 subunit of the gamma-amino butyric acid receptor (GABRA3), a candidate gene for BP illness located in this region. The genetic data were analyzed with the LOD score method using age-dependent penetrance of an autosomal dominant disease gene and narrow and broad clinical models. In MAD3 and MAD4 the multipoint LOD score data suggested a localization of a BPI gene again near F9. In the 7 new families the overall linkage data excluded the Xq27-q28 region. However, if the families were grouped according to their proband's phenotype BPI or BPII, a susceptibility gene for BPI disorder at the DXS52-F8 cluster could not be excluded.

Captopril renography and the effect of percutaneous transluminal angioplasty on blood pressure in 94 patients with renal artery stenosis.

We treated 94 patients by percutaneous transluminal angioplasty (PTA) for renal artery stenosis (RAS). Prior to PTA, a renogram during angiotensin converting enzyme (ACE) inhibition with captopril was performed, but the result did not influence the decision to treat. The parenchymal time activity curves were judged by visual interpretation. Of the 94 patients, 51 had unilateral and 43 bilateral RAS. In 17 patients with bilateral RAS, PTA could be performed only in the least affected kidney; because of this the effect of PTA on their blood pressure could not be evaluated. Of the remaining 77 patients, a positive captopril renogram was seen in all 31 cured patients, in 22 of the 27 patients with improvement, and in six of the 19 patients with no change of their blood pressure. The sensitivity of the tests for cure and improvement of the blood pressure was 91% (53/58 patients) for all patients, 95% in patients with unilateral RAS (35/37), and 86% (18/21 patients) in patients with bilateral RAS, bilaterally treated. In 18 patients with a negative captopril renogram the blood pressure improved in five, and did not change in 13 patients. The success of PTA in patients with a negative captopril renogram was so poor that we feel it would have been better not to have performed angiography and PTA at all. In conclusion, captopril renography is a useful investigation in assessing the likelihood of blood pressure reduction after PTA of renal artery stenosis.

A linkage study between bipolar disorder and genes involved in dopaminergic and GABAergic neurotransmission.

Neurobiological studies indicate a dysregulation of the dopaminergic and GABAergic neurotransmission in bipolar disorder. We examined two large families segregating bipolar disorder, for linkage with the genes encoding dopamine beta-hydroxylase, the dopamine transporter DAT1, the dopamine D2, D3 and D5 receptors, and the alpha-1, alpha-5 and beta-1 subunits of the GABAA receptor. Under at least one diagnostic model one of the two families provided evidence to exclude linkage for the DAT1, DRD2, DRD3, DRD5, DBH, GABRA1 and GABARB1 genes but could not exclude the GABRA5 locus. A second family excluded only the GABRA1 and GABRA5 loci at zero recombination and could not formally reject linkage at the DBH, DRD2, DRD3, DRD5, DAT1 and GABARB1 loci. Further analyses at these loci are warranted.

Quantitation of trans-vitamin K1 in small serum samples by off-line multidimensional liquid chromatography.

A sensitive high-performance liquid chromatography method for vitamin K1 using normal-phase fractionation was updated in order to cope with the limited volume of blood samples obtained from newborns. The method combines sample clean-up, normal-phase fractionation with reversed-phase chromatography using post-column electrochemical reduction and fluorescence detection. It was possible to measure vitamin K1 concentration in at least 0.2 ml of (potentially lipemic) serum even at a level of 0.3 nmol/l. The normal-phase method was compared to the updated method. The updated method shows about a twenty-fold lower limit of detectability than the previously described method.

Vitamin K1 in the newborn.

By a sensitive high-performance liquid chromatography method it was possible to measure vitamin K1 concentrations in mothers and their newborn children, even at a level of 0.5 nmol/l. All neonates were born after an uncomplicated and full-term pregnancy. The vitamin K1 concentration in neonates proved to be about half the value of their mothers, which is consistent with earlier studies of vitamin K1 dependent clotting factors. One may assume the existence of a partial placental barrier to vitamin K1. Venous, arterial and mixed cord blood did not differ significantly, so puncturing the vena umbilicalis is sufficient to measure vitamin K1 concentrations in cord blood.