Parous women perform less moderate to vigorous physical activity than their nulliparous peers: a population-based study in Denmark.

OBJECTIVES: The World Health Organization (WHO) highlights parous women as a key population for monitoring trends of physical activity (PA). We aimed to estimate the proportion of Danish women non-adhering to WHO PA guidelines in parous women compared with nulliparous women and to describe leisure-time PA intensity in each of these groups. STUDY DESIGN: Cross-sectional study. METHODS: This population-based study builds on a sample of 27,668 women aged 16-40 years from the Danish National Health Survey 2021. These data were linked with childbirth data from the Danish National Birth Registry. The primary outcome was self-reported weekly hours of moderate to vigorous leisure-time PA (MVPA) dichotomized into: (i) adhering to WHO guidelines for MVPA or (ii) not adhering to WHO guidelines for MVPA. Binomial regression analysis was used to calculate prevalence proportions (PP) and prevalence proportion ratios (PPR). RESULTS: Of the 27,668 women, a total of 20,022 were included; 9338 (46.6%) parous women and 10,684 (53.4%) nulliparous women. The PP of women non-adhering to WHO PA guidelines was 63.8% (95% CI 62.9-64.8) for parous and 51.3% (95% CI 50.4-52.3) for nulliparous women, corresponding to a PPR of 1.24 (95% CI 1.21; 1.27). CONCLUSIONS: The proportion of parous women who did not adhere to WHO PA guidelines for MVPA was 24% higher than that of nulliparous women. This highlights parous women as a subgroup of the adult population at increased risk of non-adherence to WHO PA guidelines. These findings call for future research to inform new strategies aiming to promote PA in parous women.

Reproducibility and responsiveness of a Danish Pedi-IKDC subjective knee form for children with knee disorders.

The modified International Knee Documentation Committee Subjective Knee Form (Pedi-IKDC) is a widely used patient-reported tool ranging on a scale from 0 to 100. We aimed to translate Pedi-IKDC into Danish and assess its reproducibility and responsiveness in children with knee disorders. The translation complied with the international guidelines. Reproducibility was assessed in 53 children (15 years) responding Pedi-IKDC at baseline and after 3-14 days. For analysis of responsiveness, 94 children (15 years) responded Pedi-IKDC again after 3 months. Test-retest reliability was excellent. Intraclass correlation coefficient was 0.9, standard error of measurement was 4.1 points, and smallest detectable change (SDC) was 11.3 points. Evaluating responsiveness as a large effect was found in children reporting improvement compared with children reporting deterioration. The change score was correlated to the external anchor Global Rating Scale consisting of 15 answers from -7 "A very great deal worse" to +7 "A very great deal better," with a Spearmen's rho of 0.45 (P > 0.001). The minimal clinically important changes was 12.0. In conclusion, excellent test-retest reproducibility was found at group level, but at individual level the SDC was high. The Pedi-IKDC showed adequate responsiveness and is suitable for assessing improvement or deterioration in children with knee disorders.

Eccentric hip abductor weakness in patients with symptomatic external snapping hip.

Symptomatic external snapping hip can be a long-standing condition affecting physical function in younger people between 15-40 years. Gluteal weakness has been suggested to be associated with the condition. The aim of this study was to investigate whether eccentric hip abduction strength is decreased in patients with external snapping hip compared with healthy matched controls, and to examine isometric hip abduction, adduction, extension, flexion, internal rotation, and external rotation in patients with external snapping hip and matched controls. Thirteen patients with external snapping hip were compared with 13 healthy matched controls in a cross-sectional study design. The mean age of the patients was 25.5 ± 3.4 years and the mean age of the controls was 25.6 ± 2.6 years. Eccentric and isometric strength were assessed with a handheld dynamometer, using reliable test procedures. Eccentric hip abduction strength was 16% lower in patients with external snapping hip compared with healthy matched controls (1.50 ± 0.47 Nm/kg versus 1.82 ± 0.48 Nm/kg, P = 0.01). No other strength differences were measured between patients and controls (P > 0.05). Eccentric hip abductor weakness was present in patients with symptomatic external snapping hip compared with healthy matched controls.

Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position.

In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B1-2,3-dichloride and transfected into appropriate Escherichia coli cells. Forward mutations in the lacZ alpha-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing. Transfection of modified DNA into uvrA-/mucAB+ cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction. Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic. A majority of point mutations under all conditions affected G:C bp. Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells. Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions. Point mutations occurred predominantly at predicted damage hotspots. The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens. The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion. In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion.

Mechanisms of mutagenesis by chloroacetaldehyde.

A number of bifunctional chemical mutagens induce exocyclic DNA lesions. For example, 2-chloroacetaldehyde (CAA), a metabolite of vinyl chloride, readily reacts with single-stranded DNA to predominantly form etheno lesions. Here, we report on in vivo mutagenesis caused by CAA treatment of DNA in vitro. These experiments used partially duplex phage M13AB28 replicative form DNA in which a part of the lacZ gene sequence was held in single-stranded form to direct reaction with CAA. CAA-treated partial duplex DNA was transfected into Escherichia coli, and the induced base changes were defined by DNA sequencing. These experiments suggested that CAA treatment induced mutations at cytosines, much less efficiently at adenines, but not at guanines or thymines. Among mutations targeted to cytosine, 80% were C-to-T transitions and 20% were C-to-A transversions. Application of a post-labeling method detected dose-dependent formation of ethenoadenine and ethenocytosine in CAA treated DNA. These data indicate that ethenocytosine is a highly efficient mutagen with properties suggestive of a non-instructional DNA lesion in vivo. Paradoxically, ethenoadenines are efficiently bypassed by a mechanism which appears to be largely nonmutagenic.

Quantitative measurement of alternatively spliced amyloid precursor protein mRNA expression in Alzheimer's disease and normal brain by S1 nuclease protection analysis.

We have used an S1 nuclease protection strategy to measure alternatively spliced amyloid precursor protein (APP) mRNAs associated with Alzheimer's disease (AD) to determine whether the expression of either one or more of the transcripts correlate with observed amyloid plaque pathology. Comparison of AD with normal cortex reveals that increasing plaque density parallels an increase in the fraction of APP-695 and a corresponding decrease in APP-770 and 751 mRNA fractions. A specific increase of APP-695, the protease inhibitor-lacking APP RNA form, in those brain regions most involved with amyloid plaque formation, suggests that an imbalance in the protease inhibitor is potentially significant in the disease. These data are consistent with cellular/tissue region-specific regulation of alternative splicing accounting for AD-related changes in the expression of APP mRNA forms.

A novel species-specific RNA related to alternatively spliced amyloid precursor protein mRNAs.

Using an S1 nuclease protection assay, we have identified a novel "variant" Amyloid Precursor Protein (APP) RNA in human brain which is 3-6-fold more abundant than APP-770, but less abundant than APP-751 or APP-695. This variant, referred to as amyloid precursor-related protein 365 (APRP-365), is not detected in mouse and rat brain RNAs. A 1.6 kilo-basepair cDNA clone corresponding to this variant APP RNA predicts the existence of a 365 amino acid protein that is similar to the amino-terminal end of APP-770 but lacks the beta-amyloid peptide and any hydrophobic transmembrane spanning domains. In a modified polymerase chain reaction (PCR), we used amplification of reverse transcribed mRNA to confirm and extend our S1 observations. Together, the features of APRP-365 suggest that the human variant is a soluble protein containing a Kunitz protease inhibitor domain.

Altered binding of mutated presenilin with cytoskeleton-interacting proteins.

The majority of familial Alzheimer's disease (AD) cases are linked to mutations on presenilin 1 and 2 genes (PS1 and PS2). The normal function of the proteins and the mechanisms underlying early-onset AD are currently unknown. To address this, we screened an expression library for proteins that bind differentially to the wild-type PS1 and mutant in the large cytoplasmic loop (PS1L). Thus we isolated the C-terminal tail of the 170 kDa cytoplasmic linker protein (CLIP-170) and Reed-Sternberg cells of Hodgkin's disease-expressed intermediate filament-associated protein (Restin), cytoplasmic proteins linking vesicles to the cytoskeleton. PS1L binding to CLIP-170/restin requires Ca(2+). Treating cells with thapsigargin or ionomycin increased the mutated PS1 in CLIP-170 immunoprecipitates. Further, PS1 and CLIP-170 co-localize in transfected cells and neuronal cultures.

Effect of increased glycogen synthase kinase-3 activity upon the maturation of the amyloid precursor protein in transfected cells.

Aberrant control of protein phosphorylation is an important feature in Alzheimer's disease pathology. The action of glycogen synthase kinase-3 beta (GSK-3 beta) on the maturation and phosphorylation of an amyloid precursor protein-reporter construct (APP-REP) was studied in transfected COS-7 cells. Elevation of GSK-3 beta activity by enzyme over-expression resulted in an increase in the level of mature forms of co-expressed APP-REP. This effect was not associated with an increased level of APP-REP phosphorylation at Thr743, an in vitro GSK-3 beta phosphorylation site. These findings suggest that GSK-3 beta activity may indirectly increase cellular maturation of APP, which may subsequently result in altered production of beta-amyloid protein.

DNA replication-blocking properties of adducts formed by aflatoxin B1-2,3-dichloride and aflatoxin B1-2,3-oxide.

The carcinogen aflatoxin B1 (AFB1), upon activation to a hypothesized AFB1-2,3-oxide (AFB1-oxide), reacts with DNA guanines. Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was originally synthesized as an electronic analog for the putative AFB1-oxide, which has never been isolated due to presumed reactivity. We have previously shown that AFB1-oxide reacts with base-paired DNA guanines in a sequence-specific manner, as revealed by an alkali-degradation analysis. On the basis of a replication-block analysis, we have shown that AFB1-Cl2 reacts with single-stranded DNA preferentially at inverted repeat sequences, which were suggested to be capable of forming intrastrand base-paired structures. Here, we present data to show the following. Both AFB1-oxide and AFB1-Cl2 react with guanines in double-stranded DNA to induce similar sequence-specific, alkali-labile sites. Reactivity with partial DNA duplexes as well as the use of single-strand specific chemical probes directly demonstrates that AFB1-Cl2, like AFB1-oxide, prefers base-paired guanines over non-base-paired guanines. DNA replication block patterns induced by AFB1-oxide are essentially similar to those induced by AFB1-Cl2. Unexpectedly, and unlike other tested DNA lesions, Mn2+ does not appear to affect the template blocking properties of the adduct formed by AFB1-Cl2 or AFB1-oxide. The sites for replication stoppage as well as the lack of a Mn2+ effect on adducted templates have implications for the mechanisms of mutagenesis by activated AFB1.

Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies.

Melanoma-associated antigens (MAAs) recognized by murine monoclonal and rabbit polyclonal antibodies were compared. Two rabbit antimelanoma sera raised in our laboratory recognized five human cell-surface MAAs with approximate MWs of 75, 95, 120, 150, and 240 kd. These antigens were easily detected by SDS-PAGE of specific immunoprecipitates on a melanoma cell (HM31) which failed to reveal antigens reactive with the panel of murine monoclonal antibodies studied. By contrast, these five antigens could not be detected on another melanoma cell (SK-MEL-28), which was reactive with several of the murine monoclonals. These results suggest that the MAAs recognized by rabbit and murine antibodies are different and imply that the antigens which are immunogeneic in man may not necessarily be immunogeneic in mice.

Normative values of eccentric hip abduction strength in novice runners: an equation adjusting for age and gender.

PURPOSE: Low eccentric strength of the hip abductors, might increase the risk of patellofemoral pain syndrome and iliotibial band syndrome in runners. No normative values for maximal eccentric hip abduction strength have been established. Therefore the purpose of this study was to establish normative values of maximal eccentric hip abduction strength in novice runners. METHODS: Novice healthy runners (n = 831) were recruited through advertisements at a hospital and a university. Maximal eccentric hip abduction strength was measured with a hand-held dynamometer. The demographic variables associated with maximal eccentric hip abduction strength from a univariate analysis were included in a multivariate linear regression model. Based on the results from the regression model, a regression equation for normative hip abduction strength is presented. RESULTS: A SIGNIFICANT DIFFERENCE IN MAXIMAL ECCENTRIC HIP ABDUCTION STRENGTH WAS FOUND BETWEEN MALES AND FEMALES: 1.62 ± 0.38 Nm/kg (SD) for males versus 1.41 ± 0.33 Nm/kg (SD) for females (p < 0.001). Age was associated with maximal eccentric hip abduction strength: per one year increase in age a -0.0045 ± 0.0013 Nm/kg (SD) decrease in strength was found, p < 0.001. Normative values were identified using a regression equation adjusting for age and gender. Based on this, the equation to calculate normative values for relative eccentric hip abduction strength became: (1.600 + (age * -0.005) + (gender (1 = male / 0 = female) * 0.215) ± 1 or 2 * 0.354) Nm/kg. CONCLUSION: Normative values for maximal eccentric hip abduction strength in novice runners can be calculated by taking into account the differences in strength across genders and the decline in strength that occurs with increasing age. Age and gender were associated with maximal eccentric hip abduction strength in novice runners, and these variables should be taken into account when evaluating eccentric hip abduction strength in this group of athletes. LEVEL OF EVIDENCE: 2A.

Chloroperbenzoic acid induced DNA damage and peracid activation of alfatoxin B1.

The activated form of aflatoxin B1 (AFB1) in vitro and in vitro is believed to be the 2,3-oxide, which cannot be isolated, presumably due to its reactivity. In addition to in vitro activation by crude metabolic enzymes, two chemical procedures are available, one involving oxidation with the mild organic oxidant chloroperbenzoic acid and another involving the synthesis of AFB1-2,3-dichloride, an electronic analog of AFB1-2,3-oxide. Here we show that chloroperbenzoic acid by itself can modify DNA, primarily at non-basepaired adenine and guanine residues, as revealed by 'replication block' analysis.

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro.

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of ochratoxin A in Danish wheat and rye, 1992-99.

Ochratoxin A concentrations in rye and wheat in Denmark for 1992-99 are reported. The results show that the concentration of ochratoxin A is higher in rye than in wheat for both conventionally and organically grown rye and wheat. The levels in organically grown rye are higher than in conventionally grown based on multiyear mean contents. However, the difference between the two groups of cereals has decreased since the Danish food-monitoring system for ochratoxin A was started in 1986; 2.0% of all samples exceeded the Danish maximum limit of 5 micro g kg(-1) introduced in 1995. For rye samples, 3.2% exceeded the maximum limit, and for wheat samples, 0.5% exceeded the maximum limit.

Sequence context effects in DNA replication blocks induced by aflatoxin B1.

The genotoxic effects of the potent mutagenic carcinogen aflatoxin B1 (AFB1) are believed to be mediated by its reaction with the N-7 atom of guanine residues in DNA. We have analyzed the effect of AFB1-induced chemical modification on the template function of single-stranded DNA in vitro. The experimental strategy involves the elongation of a primer on a modified template by Escherichia coli DNA polymerase I (large fragment) and analysis of the products by high-resolution gel electrophoresis. Our data show that (i) AFB1 induces specific replication blocks one nucleotide 3' to the sites of occurrence of guanine residues on template DNA; (ii) AFB1-induced replication blocks occur predominantly at sequences capable of participation in intrastrand base pairing; (iii) within the intrastrand base-paired regions there are strong sequence context effects, in accordance with the previously described [Muench, K. F., Misra, R. P. & Humayun, M. Z. (1983) Proc. Natl. Acad. Sci. USA 80, 6-10] specificity "rules" that apply to the reaction of AFB1 with guanine residues in double-stranded DNA; (iv) there is evidence that the (7-guanyl)-AFB1 adducts as well as secondary derivatives such as the formamidopyrimidine-AFB1 act as replication blocks. In summary, these data suggest that previously observed inhibition of DNA replication and transcription by AFB1 is directly attributable to (7-guanyl)-AFB1 adducts or their secondary reaction products.

Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells.

Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.

Isolation and expression of multiple forms of beta amyloid protein precursor cDNAs.

Alzheimer's disease (AD) is associated with the extra-normal accumulation of a 42 amino acid (aa) beta-amyloid peptide (BAP) in amyloid plaques and cerebrovascular deposits. Though BAP is deposited exclusively in brains of AD and Down syndrome patients, the mRNA encoding the putative precursor to BAP is found in the brain and in peripheral tissues. Using an HL 60 cDNA library, we have isolated two Amyloid Peptide Precursor (APP) cDNAs with sequences that code for proteins containing 751 and 770 aa (APP 751 and APP 770). These longer forms of APP encode a novel region of 56 aa which is homologous to the Kunitz domain of serine protease inhibitors which is not found in the 695 aa form (APP 695) isolated from brain (1). We have examined APP expression at the RNA level using Northern blots and S1 nuclease protection studies in which the lengths, distributions and relative abundances of APP RNAs were assayed. We find that brain, WA 17 cells and NG 108-15 cells contain all three forms of APP RNAs while HL 60 cells, TMT3 cells and AB-1 cells contain predominantly the APP 751 and APP 770 RNAs.

Biotinylated and cysteine-modified peptides as useful reagents for studying the inhibition of cathepsin G.

An assay for studying the proteolytic activity of endopeptidases using a biotinylated and cysteine-modified peptide has been developed. This assay is rapid, sensitive, and reproducible. Although used here specifically for the enzyme which cleaves at the amino terminus (N-terminus) of beta-amyloid peptide (BAP); this type of radiolabeled substrate is readily applied to the analysis and detection of other endoprotease activities. This method relies on a peptide substrate which contains: (a) the amino acids flanking the enzymatic cleavage site, (b) an added cysteine at the carboxy-terminus to allow for incorporation of radiolabel via an addition reaction with tritiated N-[ethyl-1,2-3H]maleimide (3H-NEM), and (c) a biotin at the N-terminus to allow for binding to avidin-coated scintillation proximity assay (SPA) beads. It has been suggested that the enzyme involved in the N-terminal cleavage of amyloid precursor peptide to generate BAP is a chymotrypsin-like serine protease such as cathepsin G. To study this enzymatic activity and to screen for its inhibitors, we have synthesized the peptide biotin-SEVKMDAEFdC which contains the amino acids flanking the N-terminal cleavage site of BAP. Tritiated NEM is covalently bound to the cysteine at the carboxy-terminal end and the labeled peptide is purified by reverse-phase high-performance liquid chromatography. Following digestion of 3H-NEM-labeled peptide by cathepsin G, the biotinylated side of the cleaved peptide is bound to the SPA bead, while the tritiated end of the cleaved peptide remains in solution. Enzymatic hydrolysis is measured as the loss of 3H-induced scintillation signal. This method has allowed us to rapidly determine kinetic constants and develop a high throughput screen to study inhibition of cathepsin G cleavage in a native peptide context.