A New Stepwise Molecular Work-Up After Chorionic Villi Sampling in Women With an Early Pregnancy Loss.

Objective: To explore the use of a new molecular work-up based on the stepwise use of Quantitative Fluorescence PCR (QF-PCR) extended to eight chromosomes and single nucleotide polymorphism array (SNP-array) in chorionic villi obtained by chorionic villi sampling (CVS) offered to women experiencing an early pregnancy loss. Methods: During a 3-year period (January 2016-December 2018), CVS was offered to women experiencing an early pregnancy loss before the evacuation of the products of conception (POC) to retrieve chorionic villi, irrespective of the number of previous losses. A new molecular work-up was prospectively assayed encompassing a first QF-PCR round (with the 21, 18, 13, 7, X, and Y chromosomes), a second QF-PCR round (with the 15, 16, and 22 chromosomes), and a high resolution SNP-array in those cases with normal QF-PCR results. A control group in which POC were collected after surgical uterine evacuation was used to be compared with the intervention group. Results: Around 459 women were enrolled in the intervention group (CVS) and 185 in the control group (POC after uterine evacuation). The QF-PCR testing success rates were significantly higher in the intervention group (98.5%: 452/459) as compared to the control group (74%: 109/147; p < 0.001), while the chromosomal anomaly rate at the two QF-PCR rounds was similar between the two groups: 52% (234/452) in the intervention and 42% (46/109) in the control group (p = 0.073). The SNP-array was performed in 202 QF-PCR normal samples of the intervention group and revealed 67 (33%) atypical chromosomal anomalies (>10 Mb), 5 (2.5%) submicroscopic pathogenic copy number variants, and 2 (1%) variant of uncertain significance (VOUS). Conclusion: Eighty-two percent of women experiencing an early pregnancy loss opted for a CVS. The testing success rates were higher in the intervention group (CVS; 98%) as compared to the control group (POC; 74%). The overall yields were 52% by QF-PCR (including three complete hydatiform moles), and 16% by SNP-array, including 15% atypical chromosomal anomalies and 1.1% submicroscopic pathogenic copy number variants.

Is Cisterna Magna Width a Useful First-Trimester Marker of Aneuploidy?

OBJECTIVE: To assess whether the cisterna magna (CM) width measured in first-trimester fetuses is a useful marker for aneuploidy detection. METHODS: This was a prospective study in 2 different cohorts in a tertiary referral center. The first cohort comprised 913 fetuses from the general pregnancy population during the period 2012-2016 and was used to construct the CM reference ranges applying the λ-µ-σ (LMS) method. The second cohort included 714 high-risk fetuses undergoing chorionic villus sampling during the period 2012-2016. Mean detection rates using the 95th percentile for CM width observed in chromosomal anomaly groups were compared with those obtained in chromosomally normal fetuses. RESULTS: The 50th percentile for CM ranged from 1.66 to 2.75 mm when crown-rump length (CRL) increased from 45 to 84 mm. Among high-risk fetuses, the following chromosomal anomalies were diagnosed in 125 (17%) fetuses: trisomy 21 (n = 63), trisomy 18 or 13 (n = 21), monosomy X (n = 9), submicroscopic anomalies (n = 11), and other anomalies (n = 22). The mean CM width for euploid fetuses was 2.4 mm (1.13 multiples of the median, MoM). While CM width was significantly increased in trisomy 21 (mean 2.7 mm; 1.23 MoM; p > 0.05), no differences were found in the other anomaly groups. Among the 63 fetuses with trisomy 21, a CM width above the 99th percentile was observed in 23 fetuses (37%). CONCLUSIONS: The new reference range for CM width at 11-13 weeks of gestation did not differ from previous studies. In first-trimester fetuses with trisomy 21, CM width appears to be increased, although its value as an ultrasound marker is limited, because of its detection rate of 37%.

Parental Origin of the Retained X Chromosome in Monosomy X Miscarriages and Ongoing Pregnancies.

OBJECTIVE: To assess the distribution of the parental origin of the retained X chromosome in monosomy X, either in miscarriages or in ongoing pregnancies. METHOD: The parental origin of the X chromosome was determined in monosomy X pregnancies, either miscarriages or ongoing pregnancies. Microsatellite marker patterns were compared between maternal and fetal samples by quantitative fluorescence polymerase chain reaction. Distributions of maternally and paternally derived X chromosome were assessed in miscarriages and in ongoing pregnancies using two-tailed Fisher exact test. RESULTS: Forty monosomy X pregnancies were included in the study: 26 miscarried at 5-16 weeks, and 14 ongoing pregnancies were diagnosed at 11-20 weeks. The retained X chromosome was maternally derived in 67% of the cases. In miscarriages, maternal and paternal X chromosome were retained in a similar proportion (54% [95% CI: 35-73%] vs. 46% [95% CI: 27-65%]), while in ongoing pregnancies, the maternal rate was 13 times higher (93% [95% CI: 79-100%)] vs. 7% [95% CI: 0-20%]). CONCLUSIONS: The retained X chromosome in individuals with monosomy X should theoretically be maternally derived in 2/3 of the cases. Our study suggests a preferential early miscarriage in pregnancies with a retained paternally derived X chromosome. This may explain the observation that 75-90% of individuals with monosomy X retain the maternal X chromosome.

Chromosomal Microarray Analysis in Fetuses with Growth Restriction and Normal Karyotype: A Systematic Review and Meta-Analysis.

OBJECTIVE: To perform a systematic review of the literature and a meta-analysis to estimate the incremental yield of chromosomal microarray analysis (CMA) over karyotyping in fetal growth restriction (FGR). METHODS: This was a systematic review conducted in accordance with the PRISMA criteria. All articles identified in PubMed, Ovid Medline, and ISI Web of Knowledge (Web of Science) from January 2009 to November 2016 describing pathogenic copy number variants (CNVs) in fetuses with growth restriction were included. Case reports were excluded. Risk differences were pooled to estimate the overall and stratified CMA incremental yield. RESULTS: Ten studies with full data available met the inclusion criteria for analysis. Combined data from these studies revealed a 4% (95% confidence interval [CI] 1-6%) incremental yield of CMA over karyotyping in nonmalformed growth-restricted fetuses, and a 10% (95% CI 6-14%) incremental yield in FGR when associated with fetal malformations. The most frequently found pathogenic CNVs were 22q11.2 duplication, Xp22.3 deletion, and 7q11.23 deletion (Williams-Beuren syndrome), particularly in isolated FGR. CONCLUSION: The use of genomic CMA provides a 4% incremental yield of detecting pathogenic CNVs in fetuses with isolated growth restriction and normal karyotype.

Cervical Alpha-Actinin-4 Is Upregulated in Women with Threatened Preterm Labor and Microbial Invasion of the Amniotic Cavity.

OBJECTIVE: To characterize the proteome profile of women with threatened preterm labor (PTL) below 34;0 weeks with and without microbial invasion of the amniotic cavity (MIAC) using mass spectrometry in the amniotic fluid (AF) and Western blot analysis in the cervical mucus and the vaginal fluid. SUBJECTS AND METHODS: In the discovery phase, a case-control study including 8 women with MIAC and 7 without matched for gestational age at sampling was performed. Proteomic profile characterization was done using the LTQ VELOS Orbitrap mass spectrometer in the AF. In the validation phase, a selection of the proteins differentially expressed by mass spectrometry in the genital samples of a prospective cohort of 109 women was validated by Western blot analysis. RESULTS: In the discovery phase, the mass spectrometry analysis identified a total of 444 proteins. Sixteen were chosen for validation, being involved in defense (calgranulin A, B, C, C-reactive protein), cytoskeletal remodeling (alpha-actinin-4 [ACTN-4], plastin-2, α2-antiplasmin, vitronectin), metabolism (cystatin-ß, glucose 6 phosphate isomerase, glutathione S-transferase, prostaglandin D2 synthase, corticosteroid-binding globulin), and vascular (α1-antichymotrypsin, hemopexin, endosialin) pathways. In the validation phase, cervical ACTN-4 was the only significantly upregulated protein in women with MIAC with an odds ratio of 6.8 (p = 0.002). CONCLUSIONS: Cervical ACTN-4 was significantly upregulated in the group of women with PTL with MIAC.

Heterotrisomy recurrence risk: a practical maternal age-dependent approach for excess trisomy 21 risk calculation after a previous autosomal trisomy.

A new maternal age-dependent method to estimate absolute excess risks of trisomy 21, either after a previous trisomy 21 (homotrisomy) or after another trisomy (heterotrisomy), is proposed to be added to the estimated risk by conventional screening methods. Excess risk at term for a subsequent trisomy 21 was calculated from midtrimester risks reported by Morris et al., decreasing from 0.49% at 20 years to 0.01% at 46 years at the index pregnancy. Excess risk after a previous uncommon trisomy was derived from data reported by Warburton et al., decreasing from 0.37% at 20 years to 0.01% at 50 years.

Genomic Microarray in Fetuses with Early Growth Restriction: A Multicenter Study.

BACKGROUND: Little information is available about the risk of microdeletion and microduplication syndromes in fetal growth restriction (FGR) with a normal karyotype. OBJECTIVE: To assess the incremental yield of genomic microarray over conventional karyotyping in fetuses with early growth restriction. STUDY DESIGN: Genomic microarray was prospectively performed in fetuses with early growth restriction defined as a fetal weight below the 3rd percentile estimated before 32 weeks of pregnancy, and a normal quantitative fluorescent polymerase chain reaction result. The incremental yield of genomic microarray was defined by the rate of fetuses presenting with a pathogenic copy number variant below 10 Mb. RESULTS: Among 133 fetuses with early FGR, a 6.8% (95% CI: 2.5-11.0) incremental yield of genomic microarray over karyotyping was observed. This incremental yield was 4.8% (95% CI: 0.2-9.3) in isolated FGR, 10% (95% CI: 0-20.7) in FGR with nonstructural anomalies, and 10.5% (95% CI: 0-24.3) in FGR with structural anomalies. CONCLUSION: Our multicenter study reveals that 6.8% of fetuses with early growth restriction present with submicroscopic anomalies after common aneuploidies were excluded. Even when FGR is observed as an isolated finding, genomic microarray analysis should be considered after or instead of karyotyping, due to its 4.8% incremental yield.

Effectiveness of ovarian age as the background risk for aneuploidy screening in an unselected pregnant population.

The aim of this study was to assess the performance of first-trimester combined screening when replacing the chronological maternal age by Anti-Müllerian hormone (AMH) and antral follicle count (AFC)-derived ovarian ages, as the background risk in trisomy risk estimation. A total of 639 pregnant women who completed first-trimester combined screening together with AMH and AFC determination were included. Trisomy risks were estimated based on three distinct 'maternal ages' as a-priori risk (chronological age, AMH- and AFC-derived ovarian age). The screening performance was assessed using three different approaches: received operator curve; detection rate and false positive rates for a fixed 1/250 threshold; and detection rates for a fixed 3% false positive rate. A non-significant trend was shown for AMH-derived age for both an increased area under the curve (0.986 versus 0.979) and an increased detection rate (from 83% to 100%) for a 1/250 risk threshold. For a 3% false-positive rate, a non-significant trend for increased detection with the use of both AMH- and AFC-derived ovarian ages was observed (from 67% to 83%). These results indicate that, although ovarian derived ages seem to potentially reflect a more precise background risk for fetal trisomies, the improvement in screening performance is only residual.

Intertwin crown-rump length discordance in the prediction of fetal anomalies, fetal loss and adverse perinatal outcome.

OBJECTIVE: To assess the risks of fetal anomalies, fetal loss and adverse perinatal outcome in a cohort of first-trimester intertwin crown-rump length (CRL) discordant twins, stratified by chorionicity and the degree of CRL discordance. METHOD: Four-hundred-and-seventy-one twin pregnancies were scanned during an 8-year period at 11-14 weeks, and those with an intertwin CRL discordance ≥10% were compared with concordant twins. Outcomes were also compared between monochorionic and dichorionic twins and between moderate (10-16%) and severe (>16%) discordance. RESULTS: Four-hundred-and-five twin pregnancies, 65 discordant and 340 concordant, were follow-up. Discordant twin pregnancies were at significant higher risk of chromosomal (OR = 11.42; 95% CI: 2.78-46.94) and structural anomalies (OR = 5.91; 95% CI: 2.25-15.54), spontaneous fetal loss (OR = 4.23; 95% CI: 1.79-10.01), birthweight discordance (OR = 2.8; 95% CI: 1.48-5.65) and small-for-gestational age (OR = 3.48; 95% CI: 1.78-6.79). Similar differences (except for birthweight discordance) were observed among dichorionic twins. Among monochorionic, increased frequencies were only seen for structural anomalies, birthweight discordance and small newborns. Severe CRL discordance presented with higher rates of structural anomalies, stillbirth, birthweight discordance and small newborns. CONCLUSION: Intertwin CRL discordance (≥10%) results in an increased risk of fetal anomalies and growth restriction that increases in severe CRL discordance (≥16%).

Role of ovarian reserve markers, antimüllerian hormone and antral follicle count, as aneuploidy markers in ongoing pregnancies and miscarriages.

OBJECTIVE: To assess the role of two ovarian reserve markers, antimüllerian hormone (AMH) and antral follicle count (AFC), as markers of the background risk for fetal trisomy. DESIGN: Prospective study. SETTING: Tertiary referral hospital. PATIENT(S): Assessment was carried out either in ongoing pregnancies or miscarriages in our center. INTERVENTION(S): AFC was assessed transvaginally during a routine (11-13 weeks) or referral scan. AMH was determined either during the first-trimester maternal serum markers assessment or in cases referred for chorionic villi sampling after the invasive procedure. MAIN OUTCOME MEASURE(S): AMH reference ranges were constructed according to maternal age, and AMH- and AFC-derived ovarian ages were compared among three different cytogenetic groups (normal karyotype, autosomal trisomies, and other chromosomal anomalies) in both ongoing pregnancies and miscarriages. RESULT(S): In autosomal trisomies, the median AFC-derived ovarian age was 3-5 years above the median maternal age. No differences were observed between AMH-derived ovarian age and maternal age. CONCLUSION(S): AFC-derived ovarian biologic age reflects a more precise background risk for fetal aneuploidy that is not observed for AMH-derived age.

Nuchal translucency thickness in the prediction of unbalanced translocations.

OBJECTIVE: The aim of this study was to assess the role of nuchal translucency (NT) in the prediction of unbalanced translocation in offspring of couples in which one of the parents is a balanced translocation carrier. MATERIAL AND METHODS: From January 1996 to December 2012, fetal NT was measured before chorionic villus sampling in 86 pregnancies referred because of parental balanced translocation. RESULTS: No significant differences in pregnancy characteristics and in NT expressed in millimetres or in multiples of the median (MoMs) were observed between the 41 fetuses with a normal karyotype [1.72 mm, 95% confidence interval (CI): 1.49-1.96; 1.14 MoM; 95% CI: 1.01-1.26], the 38 fetuses with balanced translocations (1.78 mm, 95% CI: 1.44-2.12; 1.22 MoM; 95% CI: 1.01-1.43) and the 7 fetuses with unbalanced translocations (2.21 mm, 95% CI: 1.33-3.09; 1.59 MoM; 95% CI: 0.72-2.45). The proportions of fetuses with NT above 95th centile in the three groups were 9.1% in fetuses with normal karyotype, 18.4% in balanced translocations and 28.6% in unbalanced translocations, not significantly different. CONCLUSION: Although a trend to an increased NT was observed in fetuses with unbalanced translocation, no significant differences were reached. According to our results, a normal NT evaluation should not preclude the performance of CVS in pregnancies of balanced translocation parents.

Antral follicle count as a marker of ovarian biological age to reflect the background risk of fetal aneuploidy.

STUDY QUESTION: Can antral follicle count (AFC) measured during pregnancy be used as a marker of ovarian age to assess the background risk of fetal aneuploidy? SUMMARY ANSWER: AFC was lower than expected according to maternal chronological age in trisomic pregnancies; therefore ovarian age could potentially reflect a more precise background risk of fetal aneuploidy screening. WHAT IS KNOWN ALREADY: The decline in a woman's reproductive function is determined by a decline in the ovarian follicle pool and the quality of oocytes. The quantitative status of ovarian reserve can be indirectly assessed by AFC, but the role of AFC as an aneuploidy risk marker in pregnant women has not been assessed yet. STUDY DESIGN, SIZE, DURATION: Our study comprised a prospective cohort including 1239 singleton pregnancies scanned before 14 weeks in our center during a 14-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Reference ranges for AFC were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies using the Lambda-Mu-Sigma method. The study population (n = 934) included 19 pregnancies with viable autosomal trisomies (trisomies 21, 18 and 13), 17 non-viable autosomal trisomies (other than 21, 18 or 13), 7 monosomies X, 1 sex trisomy and 3 triploidies (total n = 47 with chromosomal abnormalities). AFC in chromosomally abnormal pregnancies was plotted against the reference ranges. AFC multiple of the median was calculated according to the median AFC obtained by each year of age. MAIN RESULTS AND THE ROLE OF CHANCE: Sixty-eight percent of women carrying a pregnancy with viable trisomies and 65% with non-viable trisomies presented an AFC below the 50th percentile. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years above than median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age. LIMITATIONS, REASONS FOR CAUTION: We did not assess the intra- and inter-observer reliability, or use specific three-dimensional analysis which may have advantages over our two-dimensional study. In clinical practice, a drawback for assessing AFC during pregnancy is that transvaginal ultrasound is needed at the 11- to 13-week scan, when the transabdominal approach is used most commonly. Furthermore identifying ovaries by ultrasound during pregnancy could be challenging. WIDER IMPLICATIONS OF THE FINDINGS: Considering that AFC reflects ovarian aging, this 'ovarian biological age' could potentially reflect a more precise background risk of fetal aneuploidy. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by PI 11/00685. Instituto de Salud Carlos III. Fondo de Investigación Sanitaria. No competing interests declared.

Likelihood ratios to apply for nasal bone, ductus venosus and tricuspid flow at the 11-13 weeks' scan in down syndrome screening.

OBJECTIVE: To assess the feasibility of nasal bone (NB), ductus venosus (DV) and tricuspid flow (TF) at the 11-13 weeks' scan, calculate likelihood ratios for each of the markers and evaluate their efficacy in expanded and contingent screening strategies for Down syndrome. MATERIAL AND METHODS: NB, DV and TF were assessed in 11,261 singleton fetuses undergoing first trimester combined screening. For each marker, Down syndrome detection rate (DR), false positive rate (FPR), positive, negative and isolated likelihood ratios (PLR, NLR and iLR) were calculated. Likelihood ratios were multiplied to the combined test risk either to the entire population or to the intermediate risk group (expanded and sequential strategies, respectively). RESULTS: Down syndrome was diagnosed in 101 pregnancies. Feasibility for marker assessment ranged from 71 to 97%, DRs for isolated markers from 20 to 54% and FPRs from 1.3 to 5.3%. PLR ranged from 10 to 15, NLR from 0.5 to 0.8 and iLR from 3.9 to 5.6. When ultrasound markers were added to both strategies, a significant FPR reduction was observed. CONCLUSION: The application of NB, DV and TF likelihood ratios to the combined test risk, either in an expanded or contingent strategy, result in a FPR reduction.

First-trimester Down syndrome screening in renal-transplanted pregnant women: a model for adjusting the false-positives rates.

OBJECTIVE: This study aimed to develop a model to adjust the increased ß-hCG levels observed in renal-transplanted women, leading to increased false-positive rates in Down syndrome screening. METHODS: Detailed data from 11 renal-transplanted and a nested-cohort of 70 pregnant women, matched by age, parity and gestational age were retrieved from our hospital records. Patient's age, multiples of the median (MoM) values for freeß-hCG, pregnancy-associated plasma protein-A, nuchal translucency, and creatinine concentration and clearance were noted. Freeß-hCG levels were adjusted according to the deviation of serum creatinine concentration by means of three different methods (median, proportionality and regression). Subsequently, Down syndrome risk was estimated with the three resulting adjusted fß-hCG values. RESULTS: After adjustment, the median ß-hCG MoM decreased from 2.15 MoM to 1.00 MoM (median method), 1.61 MoM (proportionality method) or 1.16 MoM (regression method). The non-adjusted 27% false-positive rate dropped to 18% (median method) and 10% (proportionality or regression methods) after re-estimation of the Down syndrome risk. In controls, the observed median for ß-hCG MoM was 1.12, and the false-positive rate was 5.7%. CONCLUSIONS: In first-trimester Down syndrome screening, fß-hCG adjustment by the regression method appears to be the best to match with controls.

The effect of maternal age on chromosomal anomaly rate and spectrum in recurrent miscarriage.

STUDY QUESTION: Is there any effect of maternal age on chromosomal anomaly rate and spectrum in recurrent miscarriage? SUMMARY ANSWER: There was no significant difference in the chromosome abnormality rate between sporadic and recurrent miscarriage but the chromosome abnormality rate increased significantly with maternal age. WHAT IS KNOWN ALREADY: About 50-70% of non-recurrent miscarriages occur because of a chromosomal anomaly, but no agreement about the effect of either maternal age or the number of previous miscarriages on the chromosomal anomaly rate has been reached. STUDY DESIGN, SIZE, DURATION: A retrospective cohort of 353 miscarriages successfully karyotyped in the same center between 2002 and 2011, grouped according to the number of miscarriages and maternal age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 353 women, 153 were below 35 years (73 with sporadic, 48 with two and 32 with recurrent miscarriage) and 200 were 35 years or more (81 with sporadic, 55 with two and 64 with recurrent miscarriage). The chromosomal anomaly rate and the anomaly spectrum were compared between sporadic and recurrent miscarriage, within the two maternal age groups, using the chi-square test and the Bonferroni correction for all the P-values. Risk of chromosomal anomaly was estimated for maternal age, number of miscarriages and previous live births by multivariate binary logistic regression analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Sporadic and recurrent miscarriage did not show significantly different chromosomal anomaly rates (68 versus 60%) and maternal age was the only statistically significant predictor of the chromosomal anomaly risk we identified. Some trends were observed in the chromosomal anomaly spectrum when sporadic was compared with recurrent miscarriage: recurrent miscarriage exhibited a decrease in viable trisomies (37 versus 11%) and an increase in non-viable trisomies (38 versus 57%) in women >35 years, together with an increase in unbalanced structural anomalies (4.9 versus 29%) in younger women. LIMITATION, REASONS FOR CAUTION: The mixed origin of our study population, and the limited number of recurrent miscarriages, particularly in the younger group, limits statistical power to detect differences. WIDER IMPLICATIONS OF THE FINDINGS: The most commonly observed chromosomal anomaly type in recurrent miscarriage depends on maternal age: non-viable autosomal trisomies in older women and unbalanced structural anomalies in younger women. When a chromosomal anomaly is identified as the cause of miscarriage, additional maternal evaluation may be avoided. STUDY FUNDING/COMPETING INTERESTS: No competing interests declared.

Crosstalk between Nap1 protein and Cds1 checkpoint kinase to maintain chromatin integrity.

The nucleosome assembly protein Nap1 has been implicated in various cellular functions such as histone shuttling into the nucleus, nucleosome assembly, chromatin remodelling, transcriptional control and cell-cycle regulation in Saccharomyces cerevisiae. In Schizosaccharomyces pombe nap1 null mutant cells are viable but they showed a delay in the onset of mitosis which is rescued by the absence of the replication Cds1 checkpoint kinase. In contrast, the absence of the DNA-damage Chk1 checkpoint kinase is unable to rescue the delay. Moreover, the double nap1 cds1 mutant cells lose viability and cells show positive H2AX phosphorylation, suggesting that the viability of nap1-deleted cells is due to the Cds1 kinase. We also show that overexpression of Nap1 protein blocks the cell cycle in G1 phase.

Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Pattern of Kv beta subunit expression in macrophages depends upon proliferation and the mode of activation.

Voltage-dependent potassium channels (Kv) in leukocytes are involved in the immune response. In bone marrow-derived macrophages (BMDM), proliferation and activation induce delayed rectifier K+ currents, generated by Kv1.3, via transcriptional, translational, and posttranslational controls. Furthermore, modulatory Kv beta subunits coassociate with Kv alpha subunits, increasing channel diversity and function. In this study we have identified Kv beta subunits in mouse BMDM, studied their regulation during proliferation and activation, and analyzed K+ current parameters influenced by these proteins. BMDM express all isoforms of Kv beta1 (Kv beta1.1, Kv beta1.2, and Kv beta1.3) and Kv beta2 (Kv beta2.1), but not Kv beta4, the alternatively spliced murine Kv beta3 variant. M-CSF-dependent proliferation induced all Kv beta isoforms. However, LPS- and TNF-alpha-induced activation differentially regulated these subunits. Although LPS increased Kv beta1.3, reduced Kv beta1.2, and maintained Kv beta1.1 mRNA levels constant, TNF-alpha up-regulated Kv beta1.1, down-regulated Kv beta1.2, and left Kv beta1.3 expression unchanged. Moreover, in contrast to TNF-alpha, M-CSF- and LPS- up-regulated Kv beta2.1. K+ currents from M-CSF- and LPS-stimulated BMDM exhibited faster inactivation, whereas TNF-alpha increased tau values. Although in M-CSF-stimulated cells the half-inactivation voltage shifted to more positive potentials, the incubation with LPS and TNF-alpha resulted in a hyperpolarizing displacement similar to that in resting BMDM. Furthermore, activation time constants of K+ currents and the kinetics of the tail currents were different depending upon the mode of activation. Our results indicate that differential Kv beta expression modifies the electrical properties of Kv in BMDM, dependent upon proliferation and the mode of activation. This could determine physiologically appropriate surface channel complexes, allowing for greater flexibility in the precise regulation of the immune response.

Inactivation of the Cdc25 phosphatase by the stress-activated Srk1 kinase in fission yeast.

The mechanisms by which environmental stress regulates cell cycle progression are poorly understood. In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase. Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely. We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry. Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm. However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress.

KCNQ1/KCNE1 channels during germ-cell differentiation in the rat: expression associated with testis pathologies.

KCNQ1/KCNE1 channels are responsible for the Jervell-Lange-Nielsen cardiac syndrome, which is also characterized by congenital deafness. KCNQ1/KCNE1 is crucial for K+ transport in the inner ear. We show that KCNQ1 and KCNE1 are associated in testis and that their expression is closely regulated during development. Both genes were expressed in undifferentiated germ cells in 21-day-old rats and mostly confined to basal immature germ cells in adulthood. Leydig and Sertoli cells were negative. KCNQ1 and KCNE1 were also studied in various germ-cell pathologies. First, in spontaneous unilateral rat testis atrophy, hematoxylin-eosin analysis revealed massive germ-cell aplasia with only Sertoli cells and groups of interstitial Leydig cells. In these samples, KCNQ1 and KCNE1 were not expressed. In human seminoma samples characterized by a proliferation of undifferentiated germ cells, KCNQ1/KCNE1 protein levels were higher than in healthy samples. Our results demonstrate that the expression of KCNQ1 and KCNE1 is associated with early stages of spermatogenesis and with the presence of undifferentiated healthy or neoplastic germ cells. The presence of a K+ rich-fluid in the seminiferous tubule suggests that KCNQ1/KCNE1 is involved in K+ transport, probably during germ-cell development.