Cardiovascular function in 8- to 9-year-old singletons born after ART with frozen and fresh embryo transfer.

STUDY QUESTION: Do 8- to 9-year-old singletons conceived after frozen embryo transfer (FET) or fresh embryo transfer (Fresh-ET) have increased arterial stiffness compared to naturally conceived (NC) children? SUMMARY ANSWER: The process of FET or Fresh-ET is not associated with altered cardiovascular function in 8- to 9-year-old singletons, including arterial stiffness, as compared to NC children. WHAT IS KNOWN ALREADY: ART has been suggested to influence cardiovascular risk factors (i.e. endothelial dysfunction, increased arterial blood pressure and insulin resistance). It is not known if ART procedures alter arterial stiffness in singletons. STUDY DESIGN, SIZE, DURATION: A cohort study was carried out, including 8- to 9-year-old singletons conceived after FET, Fresh-ET and NC children (50 children in each group). This study was conducted between November 2018 and August 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 150 singletons were identified through the Danish IVF Registry and the Medical Birth Registry. They underwent cardiac magnetic resonance imaging (CMR) and anthropometric measurements. Parental data were collected using questionnaires. NC children were matched by sex and birth year with FET/Fresh-ET children. Exclusion criteria were congenital heart disease, maternal gestational diabetes or maternal diabetes mellitus. Our primary outcome was arterial stiffness, which is assessed from noninvasive arterial blood pressure and aortic ascendens distensibility. The secondary outcome was the pulse wave velocity of total aorta and exploratory outcomes were left ventricular ejection fraction, mean arterial pressure, cardiac output and total peripheral resistance. Measurements and analyses were performed blinded to the child group. MAIN RESULTS AND THE ROLE OF CHANCE: Aortic ascendens distensibility of children conceived after FET and Fresh-ET did not differ from NC children (mean (SD): FET 11.1 (3.6) 10-3 mmHg-1, Fresh-ET 11.8 (3.0) 10-3 mmHg-1, NC 11.4 (2.8) 10-3 mmHg-1, P > 0.05). Multivariate linear regression was performed to adjust for potential confounders (i.e. child sex and age, maternal BMI at early pregnancy and maternal educational level). Data showed no statistically significant differences between study groups and aortic ascendens distensibility. However, the fully adjusted model showed a non-significant tendency of lowered aortic ascendens distensibility in children born after FET compared to Fresh-ET (ß estimate (95% CI): -0.99 10-3 mmHg-1 (-2.20; 0.21)) and NC children (ß estimate (95% CI): -0.77 10-3 mmHg-1 (-1.98; 0.44)). Lastly, secondary and exploratory outcomes did not differ between the groups. Primary and secondary outcomes showed good intra-rater reliability. LIMITATIONS, REASONS FOR CAUTION: This study is possibly limited by potential selection bias as the participation rate was higher in the ART compared to the NC group. Also, in some variables, the study groups differed slightly from the non-participant population. The non-participant population (n = 1770) included those who were excluded, not invited to CMR scan, or declined to participate in this study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that children born after FET or Fresh-ET do not have altered cardiovascular function, including arterial stiffness. This is reassuring for the future use of ART. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Novo Nordisk Foundation (grant reference number: NNF19OC0054340) and The Research Foundation of Rigshospitalet. All authors declared no conflict of interests. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT03719703.

Methodical considerations when estimating nutrient digestibility in horses using the mobile bag technique.

Total collection of faeces is considered the golden standard for estimating apparent total tract digestibility (ATTD) in horses. However, the evaluation of individual feedstuffs is limited and determination of nutrient digestibility in different segments of the gastrointestinal tract (GIT) is excluded. The rationale for performing this study was that the mobile bag technique (MBT) can provide information on individual feedstuffs' degradation, and the use of fistulated animals does provide additionally information regarding degradation in individual segments of the GIT. Recommendations for using the MBT in ruminants are well established, but limited methodical studies have been published with horses. The objective of this study was to evaluate the MBT by comparing the ATTD with the nutrient disappearance and degradation kinetics of hay in horses. It was hypothesised that DM degradation as estimated by the MBT is equal to the ATTD of the DM. Furthermore, we hypothesised that bag size has no effect on nutrient disappearance but increasing the feed to surface area (FSA) decreases the DM disappearance. Five caecum cannulated horses were fed a hay-only diet (6.7 kg DM/day) with 14 days of adaptation followed by four consecutive days of total faeces collection. Three bag sizes (height × length × side, cm; 1.2 × 10 × 2, 3 × 4 × 2, 1 × 6 × 2) and three FSAs (10.4, 20.8 and 41.7 mg/cm2) were administrated at each meal (3 meals/day) on days 1 and 2 of the collection. Faeces were checked for bags every 6th h, the collection time was noted and the DM disappearance together with the transit time (TT) for each bag type was estimated. Dry matter disappearance from the individual bags was fitted to degradation profiles, and the effective degradability (ED) and degradation (Dt) were determined. The results of the study showed that the ATTD of DM, organic matter (OM), NDF and ADF can be predicted based on their disappearance from the mobile bags, but that ash and CP are overestimated in comparison to the ATTD. The TT for the bags was 29.2 h, and when using a mean retention time of 30 h to predict ED and Dt, it was clear that ED was underestimated, whereas Dt reflected the ATTD of DM. In conclusion, the MBT can be used to estimate the degradability of DM, OM and fibre as these nutrients resemble the ATTD. The bag size did not affect the DM disappearance, but the FSA should be kept below 20 mg/cm2 as higher levels might limit the degradation kinetics.

DIAGNOSIS OF ENDOCRINE DISEASE: Sex steroid action in adolescence: too much, too little; too early, too late.

ABSTRACT: This review aims to cover the subject of sex steroid action in adolescence. It will include situations with too little sex steroid action, as seen in for example, Turners syndrome and androgen insensitivity issues, too much sex steroid action as seen in adolescent PCOS, CAH and gynecomastia, too late sex steroid action as seen in constitutional delay of growth and puberty and too early sex steroid action as seen in precocious puberty. This review will cover the etiology, the signs and symptoms which the clinician should be attentive to, important differential diagnoses to know and be able to distinguish, long-term health and social consequences of these hormonal disorders and the course of action with regards to medical treatment in the pediatric endocrinological department and for the general practitioner. This review also covers situations with exogenous sex steroid application for therapeutic purposes in the adolescent and young adult. This includes gender-affirming therapy in the transgender child and hormone treatment of tall statured children. It gives some background information of the cause of treatment, the patient's motivation for medicating (or self-medicating), long-term consequences of exogenous sex steroid treatment and clinical outcome of this treatment.

An exploratory study of competition scores and salivary cortisol concentrations in Warmblood horses.

The main objective of this explorative study was to describe the relationship between competition scores and salivary cortisol concentrations in young horses during dressage and showjumping competitions. The study also investigated whether the diurnal rhythm of salivary cortisol concentrations was affected by competition over consecutive days compared with the home environment. Saliva samples were collected from 126 dressage horses and showjumpers in their home environment and at 3 different events. The relationship between scores given by judges at the competition and cortisol concentrations at the event was assessed. The results demonstrated that competition scores correlated positively to baseline cortisol concentrations at one of 3 events (r = 0.53, P < 0.001). Salivary cortisol concentrations followed a diurnal rhythm with the highest concentrations measured in the morning and the lowest in the evening, both at home and in the competition environment (P < 0.05). Salivary cortisol concentrations were greater during the competitions than at home (P < 0.05) except at one event where showjumpers did not increase between home and competition. Dressage horses had the highest baseline cortisol concentrations at competition, and exercise caused cortisol concentrations to increase in both showjumpers and dressage horses (P < 0.001). In conclusion, the diurnal rhythm in salivary cortisol concentrations was maintained in the novel environment. Dressage horses demonstrated greater baseline cortisol concentrations at competition than showjumpers, suggesting that they may perceive the novel environment as more stressful. Furthermore, there was no consistent relationship between baseline salivary cortisol concentrations and competition scores across the events.

The effect of feeding on CO2 production and energy expenditure in ponies measured by indirect calorimetry and the 13C-bicarbonate technique.

Energy expenditure (EE) can be estimated based on respiratory gas exchange measurements, traditionally done in respiration chambers by indirect calorimetry (IC). However, the (13)C-bicarbonate technique ((13)C-BT) might be an alternative minimal invasive method for estimation of CO(2) production and EE in the field. In this study, four Shetland ponies were used to explore the effect of feeding on CO(2) production and EE measured simultaneously by IC and (13)C-BT. The ponies were individually housed in respiration chambers and received either a single oral or intravenous (IV) bolus dose of (13)C-labelled sodium bicarbonate (NaH(13)CO(3)). The ponies were fed haylage 3 h before (T(-3)), simultaneously with (T(0)) or 3 h after (T(+3)) administration of (13)C-bicarbonate. The CO(2) produced and O(2) consumed by the ponies were measured for 6 h with both administration routes of (13)C-bicarbonate at the three different feeding times. Feeding time affected the CO(2) production (P<0.001) and O(2) consumption (P<0.001), but not the respiratory quotient (RQ) measured by IC. The recovery factor (RF) of (13)C in breath CO(2) was affected by feeding time (P<0.01) and three different RF were used in the calculation of CO(2) production measured by 13C-BT. An average RQ was used for the calculations of EE. There was no difference between IC and (13)C-BT for estimation of CO(2) production. An effect of feeding time (P<0.001) on the estimated EE was found, with higher EE when feed was offered (T(0) and T(+3)) compared with when no feed was available (T -3) during measurements. In conclusion, this study showed that feeding time affects the RF and measurements of CO(2) production and EE. This should be considered when the (13)C-BT is used in the field. IV administration of (13)C-bicarbonate is recommended in future studies with horses to avoid complex (13)C enrichment-time curves with maxima and shoulders as observed in several experiments with oral administration of (13)C-bicarbonate.

The effect of dietary carbohydrate composition on apparent total tract digestibility, feed mean retention time, nitrogen and water balance in horses.

A total of four diets with different carbohydrate composition were investigated in a 4×4 Latin square design experiment with four Norwegian Coldblooded trotter horses. The objective of the present study was to increase the fermentable fibre content and reduce the starch intake of the total ration obtained by partly substituting mature hay and barley with sugar beet pulp (SBP), a soluble fibre source. The diets investigated were hay only (HAY), hay (85% of dry matter intake (DMI)) and molassed SBP (15% of DMI) (SBP), hay (68% of DMI) and barley (32% of DMI) (BAR), and hay (68% of DMI), barley (26% of DMI) and SBP (6% of DMI) (BAR+SBP). The feeding level was 18.5, 17.3, 15.7 and 15.7 g DM/kg BW per day for the HAY, SBP, BAR and BAR+SBP diets, respectively. Each diet was fed for 18 days followed by 10 days of data collection, where apparent total tract digestibility (ATTD), total mean retention time (TMRT) of ytterbium-labelled hay, water balance, digestible energy (DE) intake and nitrogen balance were measured. An enzymatic chemical dietary fibre (DF) method was used to get detailed information on the composition and ATTD of the fibre fraction. Inclusion of SBP in the diet increased the ATTD of the constituent sugars galactose and arabinose (P<0.01). Feeding the HAY and SBP diets resulted in a lower TMRT owing to a higher DF intake than the BAR and BAR+SBP diets (P<0.01). There was no difference in water intake between HAY and SBP, but faecal dry matter was lower for HAY than the other diets (P=0.017), indicating that water was more tightly bound to fibre in the HAY diet. The diets were iso-energetic and provided enough DE and protein for light to moderate exercise for a 550 kg horse. In conclusion, this study showed that the DF intake had a larger effect on TMRT than partly substituting hay or barley with SBP, and that highly fermentable pectin-rich soluble DF from SBP maintains high nutrient utilization in horses.

Association between GH receptor polymorphism (exon 3 deletion), serum IGF1, semen quality, and reproductive hormone levels in 838 healthy young men.

INTRODUCTION: GH activity may be involved in male reproductive function. A common genetic polymorphism in the gene encoding the GH receptor (GHR) results in deletion of the entire exon 3 sequence (GHRd3 isoform). The short GHRd3/d3 isoform seems more sensitive compared with full-length receptors (GHRfl/fl). AIM: TO INVESTIGATE THE ASSOCIATIONS BETWEEN GH ACTIVITY, EVALUATED BY EXON 3 GHR POLYMORPHISM, AND SERUM IGF1 VS REPRODUCTIVE HORMONES, SEMEN QUALITY, AND PRE- AND POSTNATAL GROWTH IN HEALTHY YOUNG MALES (N=838, MEAN AGE: 19.4 years). RESULTS: Compared with GHRfl/fl homozygous individuals (n=467) GHRd3/d3 homozygous individuals (n=69) tended to have larger semen volume (3.2 (2.4-4.3) vs 3.6 (2.6-4.7) ml, P=0.053) and higher serum inhibin-B levels (208 pg/ml (158-257) vs 227 pg/ml (185-264), P=0.050). Semen quality, levels of gonadotropins, testosterone, estradiol, sex hormone-binding globulin, and IGF1 were not associated with GHRd3 genotype. A twofold increase in serum IGF1 was associated with a 13% (4-23) increase in calculated free testosterone (P=0.004). By contrast IGF1 was inversely associated with serum inhibin-B (P=0.027), but showed no associations to semen quality. GHR genotype and serum IGF1 were not associated with size at birth or final height. CONCLUSIONS: GHRd3 polymorphism seemed only to have a weak influence on male reproductive function of borderline significance. The sensitive GHRd3/d3 genotype may slightly increase testicular function, as evaluated by semen volume and levels of inhibin-B, but does not seem to influence Leydig cell steroidogenesis. GHR genotype did not influence pre- and postnatal growth.

Changes in anti-Müllerian hormone (AMH) throughout the life span: a population-based study of 1027 healthy males from birth (cord blood) to the age of 69 years.

CONTEXT: Anti-Müllerian hormone (AMH), which is secreted by immature Sertoli cells, triggers the involution of the fetal Müllerian ducts. AMH is a testis-specific marker used for diagnosis in infants with ambiguous genitalia or bilateral cryptorchidism. AIM: The aim of the study was to describe the ontogeny of AMH secretion through life in healthy males. SETTING: This was a population-based study of healthy volunteers. PARTICIPANTS: PARTICIPANTS included 1027 healthy males from birth (cord blood) to 69 yr. A subgroup was followed up longitudinally through the infantile minipuberty [(in cord blood, and at 3 and 12 months), n=55] and another group through puberty [(biannual measurements), n=83]. MAIN OUTCOME MEASURES: Serum AMH was determined by a sensitive immunoassay. Serum testosterone, LH, and FSH were measured, and pubertal staging was performed in boys aged 6 to 20 yr (n=616). RESULTS: Serum AMH was above the detection limit in all samples with a marked variation according to age and pubertal status. The median AMH level in cord blood was 148 pmol/liter and increased significantly to the highest observed levels at 3 months (P<0.0001). AMH declined at 12 months (P<0.0001) and remained at a relatively stable level throughout childhood until puberty, when AMH declined progressively with adults exhibiting 3-4% of infant levels. CONCLUSION: Based on this extensive data set, we found detectable AMH serum levels at all ages, with the highest measured levels during infancy. At the time of puberty, AMH concentrations declined and remained relatively stable throughout adulthood. The potential physiological role of AMH and clinical applicability of AMH measurements remain to be determined.

The effect of birthweight upon insulin resistance and associated cardiovascular risk factors in adolescence is not explained by fetal growth velocity in the third trimester as measured by repeated ultrasound fetometry.

AIMS/HYPOTHESIS: Smallness for gestational age (SGA) is associated with increased risk of developing components of the metabolic syndrome. Although SGA can imply intrauterine growth restriction (IUGR), more information is required to link specific fetal growth patterns to adult outcomes. METHODS: We examined the impact of fetal growth velocity during the third trimester (FGV) vs birthweight for gestational age on early markers of the metabolic syndrome in 123 healthy men and women (mean age 17.5 years) born at term. FGV was determined by ultrasound measurements. RESULTS: After correction for confounders including current BMI, SGA was significantly associated with raised basal plasma insulin (+19% above appropriate for gestational age), homeostasis model assessment of insulin resistance (+21%), cholesterol:HDL-cholesterol ratio (+13%) and systolic BP (+4.8%) (all p < 0.05). Furthermore SGA was associated with increased fat mass (+9.6%) and trunk-fat per cent (+6.8%) and with reduced lean body mass as determined by dual-energy X-ray absorptiometry scans (-4.1% below appropriate for gestational age) (all p < 0.05). In contrast, IUGR in the third trimester was associated only with an elevated cholesterol:HDL-cholesterol ratio (+11% above not-IUGR). CONCLUSIONS/INTERPRETATION: In the present study, FGV did not explain the impact of birthweight upon the metabolic phenotype in adolescence. This suggests that fetal growth prior to the third trimester or postnatal catch-up growth plays a more important role.

The impact of maternal smoking on fetal and infant growth.

BACKGROUND: Low birth weight is associated with accelerated postnatal growth and adverse adult health outcomes. Maternal smoking is a major risk factor for low birth weight. This study aims to assess: Pre- and postnatal growth associated with maternal smoking compared to other risk factors for low birth weight. The effect of reduction of maternal smoking on growth. SUBJECTS: A cohort (n=269) followed with ultrasound measurements in the third trimester and postnatal anthropometric measurements until 6 months of age. Mothers were interviewed about their smoking habits at 18 and 28 weeks of pregnancy. RESULTS: Maternal smoking was associated with a greater reduction in birth length SDS than other causes of equally reduced birth weight (mean difference: -0.25 SDS, P=0.013). The adjustment of gestational age, based on bi-parietal diameter at an early dating scan, indicated that mothers who reduced smoking carried smaller fetuses than mothers who continued to smoke heavily (mean difference=2.6 days, P=0.012). Birth weights in these two groups were similar (P=0.87). However at 3 months of age, reduced smoking was associated with lower weight (mean difference=-0.38 SDS, P=0.045). CONCLUSIONS: Maternal smoking was associated with a reduction of linear growth, which was more marked than that of other risk factors, and which seemed to occur before the 3rd trimester. The results indicated a beneficial effect of reduction of smoking upon third trimester growth, and that the decision to reduce smoking in mid-pregnancy may be influenced by early fetal size.

Low birth weight and male reproductive function.

Scientific interest in morbidity in children born small for gestational age (SGA) has increased considerably over the last few decades. The elevated risk of cardiovascular and metabolic diseases in adulthood in individuals born SGA has been well documented, whereas data on gonadal development are limited. Prospective studies, case-control investigations and registry surveys show that impaired intrauterine growth increases the risks of congenital hypospadias, cryptorchidism and testicular cancer approximately two- to threefold. Although few studies focus on the effect of intrauterine growth on male pubertal development, testicular hormone production or sperm quality, available evidence points towards a subtle impairment of both Sertoli cell and Leydig cell function. Animal studies support the hypothesis that impaired perinatal growth restriction, depending on the timing, can affect postnatal testis size and function into adulthood. Current human data, however, are often based on highly selected hospital populations and lack precise distinctions between low birth weight, SGA, timing of growth restriction and a differentiation of catch-up growth patterns. Despite the methodological inadequacies of individual study results, the combined evidence from all data leaves little doubt that fetal growth restriction is associated with increased risk of male reproductive health problems, including hypospadias, cryptorchidism and testicular cancer.

[Acute deep venous thrombosis and PET scanning with 2-fluoro-2-deoxy-D-glucose, FDG]. / Akut dyb venøs trombose og 2-fluoro-2-deoxy-D-glukose FDG-PET-scanning.

UNLABELLED: A 30-year-old woman with a history of malignant non-Hodgkin lymphoma with a bulky tumour on the right thigh, primarily treated with high-dose chemotherapy and involving field radiation, developed a deep venous thrombosis (DVT) in her right leg two weeks after the end of therapy. Positron emission tomography with fluorine-18 flourodeoxyglucose (FDG PET) was performed to assess the response of the lymphoma to therapy. The PET scan showed several sites of pathological, high tracer uptake corresponding to residual lymphoma, but also intense activity in the right calf correlating to the deep veins located peripheral to the site of DVT. FDG PET performed two months later showed no pathological FDG uptake in this area. CONCLUSION: Acute DVT may cause pathological uptake of FDG and should therefore be considered a possible pitfall in the interpretation of FDG PET.

A moving DNA replication factory in Caulobacter crescentus.

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.

FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains.

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His(4) and Arg(6) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn(4),Tyr(6)-->His(4),Arg(6)). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.

Proteins on the move: dynamic protein localization in prokaryotes.

Despite their small size and lack of obvious intracellular structures, bacteria have a complex and dynamic intracellular organization. Recent work has shown that many proteins, and even regions of the chromosome, are localized to specific subcellular regions that can change over time, sometimes extraordinarily fast. Protein function can depend on cellular position, so the analysis of the intracellular location of a protein can be crucial for understanding its activity. Because regulatory proteins are among those that reside at specific cellular sites, it is now necessary to consider three-dimensional organization when describing the genetic networks that control bacterial cells.

Plasmid and chromosome segregation in prokaryotes.

Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning.

The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages.

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

Complementation of the radiosensitive M059J cell line.

M059J is a radiosensitive cell line established from a human glioblastoma tumor that fails to express the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, now known as PRKDC). Another cell line, M059K, established from the same tumor is radioresistant. Neither M059J nor M059K cells have been fully characterized, beyond the lack of expression of PRKDC and low expression of ATM in M059J cells. To determine whether its radiosensitive phenotype is due to a defect in the gene that encodes PRKDC, we show here that M059J cells can be complemented with the PRKDC gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. Two hybrid cell lines that retain an extra copy of PRKDC display active kinase activity and are radioresistant, demonstrating that the primary defect in M059J cells is in PRKDC. In addition, these cell lines derived from M059J cells provide us with a closer genetic match to M059J than M059K cells in studies to elucidate the function of DNA-PK.

Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain.

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.

Chromosome segregation during the prokaryotic cell division cycle.

Recent work has dramatically changed our view of chromosome segregation in bacteria. Rather than being a passive process, it involves rapid movement of parts of the circular chromosome. Several genes involved in chromosome segregation have been identified, and the analysis of their functions and intracellular localization are beginning to shed light on the mechanisms that ensure efficient chromosome segregation.