Dissecting the surgeon's personality: cross-cultural comparisons in Western Europe.

AIM: The surgeon's personality contributes to variation in surgical decision-making. Previous work on surgeon personality has largely been reserved to Anglo-Saxon studies, with limited international comparisons. In this work we built upon recent work on gastrointestinal surgeon personality and aimed to detect international variations. METHOD: Gastrointestinal surgeons from the UK and the Netherlands were invited to participate in validated personality assessments (44-item, 60-item Big Five Inventory; BFI). These encompass personality using five domains (open-mindedness, conscientiousness, extraversion, agreeableness and negative emotionality) with three subtraits each. Mean differences in domain factors were calculated between surgeon and nonsurgeon populations from normative data using independent-samples t-tests, adjusted for multiple testing. The items from the 44-item and 60-item BFI were compared between UK and Dutch surgeons and classified accordingly: identical (n = 16), analogous (n = 3), comparable (n = 12). RESULTS: UK (n = 78, 61.5% male) and Dutch (n = 280, 65% male) gastrointestinal surgeons had marked differences in the domains of open-mindedness, extraversion and agreeableness compared with national normative datasets. Moreover, although surgeons had similar levels of emotional stability, country of work influenced differences in specific BFI items. For example, Netherlands-based surgeons scored highly on questions related to sociability and organization versus UK-based surgeons who scored highly on creative imagination (p < 0.0001). CONCLUSION: In a first cross-cultural setting, we identified country-specific personality differences in gastrointestinal surgeon cohorts across domain and facet levels. Given the variation between Dutch and UK surgeons, understanding country-specific data could be useful in guiding personality research in healthcare. Furthermore, we advocate that future work adopts consensus usage of the five factor model.

Parathyroidectomy versus cinacalcet for tertiary hyperparathyroidism; a retrospective analysis.

INTRODUCTION: Tertiary hyperparathyroidism (tHPT), i.e., persistent HPT after kidney transplantation, affects 17-50% of transplant recipients. Treatment of tHPT is mandatory since persistently elevated PTH concentrations after KTx increase the risk of renal allograft dysfunction and osteoporosis. The introduction of cinacalcet in 2004 seemed to offer a medical treatment alternative to parathyroidectomy (PTx). However, the optimal management of tHPT remains unclear. METHODS: A retrospective analysis was performed on patients receiving a kidney transplantation (KT) in two academic centers in the Netherlands. Thirty patients undergoing PTx within 3 years of transplantation and 64 patients treated with cinacalcet 1 year after transplantation for tHPT were included. Primary outcomes were serum calcium and PTH concentrations 1 year after KT and after PTx. RESULTS: Serum calcium normalized in both the cinacalcet and the PTx patients. PTH concentrations remained above the upper limit of normal (median 22.0 pmol/L) 1 year after KT, but returned to within the normal range in the PTx group (median 3.7 pmol/L). Side effects of cinacalcet were difficult to assess; minor complications occurred in three patients. Re-exploration due to persistent tHPT was performed in three (10%) patients. CONCLUSION: In patients with tHPT, cinacalcet normalizes serum calcium, but does not lead to a normalization of serum PTH concentrations. In contrast, PTx leads to a normalization of both serum calcium and PTH concentrations. These findings suggest that PTx is the treatment of choice for tHPT.

Blockade of vascular endothelial growth factor receptor 2 inhibits intraplaque haemorrhage by normalization of plaque neovessels.

BACKGROUND: Plaque angiogenesis is associated with atherosclerotic lesion growth, plaque instability and negative clinical outcome. Plaque angiogenesis is a natural occurring process to fulfil the increasing demand of oxygen and nourishment of the vessel wall. However, inadequate formed, immature plaque neovessels are leaky and cause intraplaque haemorrhage. OBJECTIVE: Blockade of VEGFR2 normalizes the unbridled process of plaque neovessel formation and induces maturation of nascent vessels resulting in prevention of intraplaque haemorrhage and influx of inflammatory cells into the plaque and subsequently increases plaque stability. METHODS AND RESULTS: In human carotid and vein graft atherosclerotic lesions, leaky plaque neovessels and intraplaque haemorrhage co-localize with VEGF/VEGFR2 and angiopoietins. Using hypercholesterolaemic ApoE3*Leiden mice that received a donor caval vein interposition in the carotid artery, we demonstrate that atherosclerotic vein graft lesions at t28 are associated with hypoxia, Hif1α and Sdf1 up-regulation. Local VEGF administration results in increased plaque angiogenesis. VEGFR2 blockade in this model results in a significant 44% decrease in intraplaque haemorrhage and 80% less extravasated erythrocytes compared to controls. VEGFR2 blockade in vivo results in a 32% of reduction in vein graft size and more stable lesions with significantly reduced macrophage content (30%), and increased collagen (54%) and smooth muscle cell content (123%). Significant decreased VEGF, angiopoietin-2 and increased Connexin 40 expression levels demonstrate increased plaque neovessel maturation in the vein grafts. VEGFR2 blockade in an aortic ring assay showed increased pericyte coverage of the capillary sprouts. CONCLUSION: Inhibition of intraplaque haemorrhage by controlling neovessels maturation holds promise to improve plaque stability.

Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms.

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

Neuropsychological presentation and adaptive skills in high-functioning adolescents with visual impairment: A preliminary investigation.

Studies in infants and young children with congenital visual impairment (VI) have indicated early developmental vulnerabilities, conversely research with older children and adults have highlighted areas of cognitive strength. A minimal amount is known, however, about the possible combination of strengths and weaknesses in adolescence, and this present study therefore aims to explore the neuropsychological presentation and adaptive behavior profile in high-functioning adolescents with congenital VI. Participants completed a battery of commonly used neuropsychological measures assessing memory, executive function, and attention. The measures utilized focused on auditory neuropsychological function, because only subtests that could be completed with auditory administration were suitable for this sample. Parents completed standardized measures of adaptive behavior, executive function, and social communication. Compared to aged-based norms for normal sight, adolescents with VI demonstrated strengths in aspects of working memory and verbal memory. Furthermore, performance across the neuropsychological battery was within or above the average range for the majority of the sample. In contrast, parent-report measures indicated areas of weakness in adaptive functioning, social communication, and behavioral executive functioning. Overall, this study provides preliminary evidence that relative to fully sighted peers, high-functioning adolescents with VI present with an uneven profile of cognitive and adaptive skills, which has important implications for assessment and intervention.

Developmental dysplasia of the hip: open reduction as a risk factor for substantial osteonecrosis.

BACKGROUND: Kalamchi and MacEwen (K&M) described a four-group scheme for classifying osteonecrosis (ON) following treatment for developmental dysplasia of the hip (DDH). However, the four groups can overlap in radiographic appearance, making assessment difficult. QUESTIONS/PURPOSES: We (1) describe a simplified K&M classification; (2) determined whether the simplified classification was reliable; and (3) assessed whether differences in the type of reduction or age at reduction resulted in different degrees of ON. PATIENTS AND METHODS: We retrospectively reviewed 300 patients with DDH treated with either open or closed reduction. We included 101 of these patients (133 involved hips). Intraobserver and interobserver reliability testing of the original and our simplified classification was performed. ON occurred in 64 hips (48%). Of these, 22 had original K&M Group I disease (classified as simplified Group A), and 42 had original K&M Groups II, III, or IV disease (classified as simplified Group B). The mean age of the patients at final followup was 12.4 years (range, 6-26.3 years). RESULTS: The interobserver reliability of the simplified classification was greater than that of the K&M classification (0.51 vs 0.33, respectively). Closed reduction after skin traction resulted in a lower incidence of Group B ON than open reduction, regardless of age at reduction. CONCLUSIONS: We propose a simplified and more reliable classification of ON after DDH. With the new classification we found type of reduction (closed with traction versus open without femoral shortening) but not age influenced the risk of ON. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Ceramic-on-metal bearings in total hip replacement: whole blood metal ion levels and analysis of retrieved components.

This study reports on ceramic-on-metal (CoM) bearings in total hip replacement. Whole blood metal ion levels were measured. The median increase in chromium and cobalt at 12 months was 0.08 microg/1 and 0.22 microg/1, respectively, in CoM bearings. Comparable values for metal-on-metal (MoM) were 0.48 microg/1 and 0.32 microg/1. The chromium levels were significantly lower in CoM than in MoM bearings (p = 0.02). The cobalt levels were lower, but the difference was not significant. Examination of two explanted ceramic heads revealed areas of thin metal transfer. CoM bearings (one explanted head and acetabular component, one explanted head and new acetabular component, and three new heads and acetabular components) were tested in a hip joint simulator. The explanted head and acetabular component had higher bedding-in. However, after one million cycles all the wear rates were the same and an order of magnitude less than that reported for MoM bearings. There were four outliers in each clinical group, primarily related to component malposition.

Natural killer cells and CD4+ T-cells modulate collateral artery development.

OBJECTIVE: The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. METHODS AND RESULTS: Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in J alpha281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II-deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. CONCLUSIONS: These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Anti-MCP-1 gene therapy inhibits vascular smooth muscle cells proliferation and attenuates vein graft thickening both in vitro and in vivo.

OBJECTIVE: Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease. METHODS AND RESULTS: MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures. CONCLUSIONS: These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Complication registration in patients after peripheral arterial bypass surgery.

The objective of this study was to describe the kinds of complications and their incidence after peripheral vascular surgery of the lower limb, coding for causes and effect on the patient. In this prospective study, a standardized complication registration system was used at the Red Cross Hospital, The Hague. All patients (n = 373) receiving an infrainguinal bypass graft during the period January 1, 1996 to December 31, 1999 were included. All postoperative complications occurring during admission were coded. In 29% of the patients 153 complications were coded. Early occlusions of the graft occurred 36 times, wound infections 21 times and postoperative hemorrhages 20 times. Postoperative retention of urine was seen most frequently of all nonspecific complications (n = 22). In 43 cases the patient needed medication or a blood transfusion for his complication. In 42 cases a re-intervention was necessary. Complications led to a prolonged stay in the hospital in 20 cases. Six patients died during admittance (mortality 1.6%). An error in surgical therapy and error in nonsurgical therapy were the cause of the complication in 108 cases (out of 153). The advantage of this complication registration is that it describes all complications, not just the specific ones. Furthermore, by categorizing all complications we force ourselves to look for errors in nonsurgical therapy and surgical technique and to describe the effect of the complication.

Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein-Barr virus.

Epstein-Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP-dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co-immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.

Incidence, impact on survival, and risk factors for multi-organ system failure in children following liver transplantation.

Liver transplantation (LTx) in children currently offers long-term survival rates of more than 80%. Many causes for Tx failure have been identified. However, the incidence and impact of multi-organ system failure (MOSF) are, to date, unknown. Therefore, in this study the role of MOSF after LTx in children was investigated with regard to outcome. The data of 114 children (53 girls, 61 boys; median age 4.3 yr) after first LTx were evaluated retrospectively. The definition of MOSF, as used by Wilkinson et al. [Crit Care Med 1986: 14: 271-274], was modified with regard to age-adjusted values. The influence of MOSF on patient survival was investigated by Kaplan-Meier analysis and multivariate regression analysis. Thirty-one of 114 children with orthotopic LTx developed MOSF (involving two or more organs). In total, 18 children died (15.8%) during the hospitalization; 16 of these had MOSF. Mortality related to two-organ failure was 29.4% (n = 5), to three-organ failure 78% (n = 7), and to four-organ failure 80% (n = 4). The highest mortality rates were observed in children with central nervous system (CNS) and cardiovascular failure, leading to a decreased probability of survival of 0.40 (p < 0.0001). Multi-variate analysis showed that CNS and cardiovascular failure were the most important risk factors for survival (p < 0.0001 and 0.056, respectively). Respiratory and renal failure, in univariate analysis, were significant contributors to poor survival, but had no statistically significant influence on outcome in multivariate analysis. Bone marrow insufficiency was found to have no influence on survival in either analysis. In multivariate analysis, the risk of development of MOSF was significantly increased by high numbers of transfused units of fresh-frozen plasma (FFP), the absence of rejection episodes, or a high bilirubin level prior to Tx. Hence, MOSF is a major factor contributing to the death of children early after LTx. CNS and cardiovascular failure carried the highest risk for a poor outcome. Other risk factors associated with the development of MOSF were: numbers of transfused units of FFP, absence of rejection episodes, and a high pre-Tx bilirubin level.

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.

Male germ cell transplantation: an experimental approach with a clinical perspective.

Germ cell transplantation was developed in strains of mice. The infusion of germ cell preparations into the seminiferous tubules of infertile hosts led to repopulation of the testis with donor germ cells and restored fertility. Meanwhile, this technique has become a powerful tool to study the expansion of the testicular stem cell population and the kinetics of spermatogonial proliferation and re-induction of spermatogenesis. Further approaches, such as the transfer of rat/hamster/rabbit germ cells into mouse testes or cryopreservation or culture of spermatogonia, have widened the spectrum of applications associated with germ cell transplantation. Since the devastating effects of chemo- or radiotherapy on spermatogonia are the cause of infertility in oncological patients, germ cell transplantation might become an alternative approach for gonadal protection in prepubertal and postpubertal male tumour patients. This review focusses on recent developments with respect to germ cell transplantation and highlights the problems and perspectives of future clinical applications.

Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples.

The measurement of serum FSH is useful in the diagnostic workup of the infertile male, but fails to predict the presence of sperm in testicular tissue. We investigated whether inhibin B reflects testicular morphology and the presence of sperm more accurately than FSH. Serum inhibin B and gonadotropin levels were determined in 91 infertile men undergoing diagnostic bilateral testicular biopsy. In 52 of the 91 patients multiple samples were taken for testicular sperm extraction (TESE). Inhibin B levels were (mean +/- SEM) 238+/-32 pg/mL in men with normal spermatogenesis (n = 9), 102+/-18 pg/mL in men with spermatogenetic arrest (n = 15), 98+/-16 pg/mL in hypospermatogenesis (n = 23), 41+/-6 pg/mL in focal Sertoli cell-only syndrome (SCO; n = 26), and 27+/-8 pg/mL in complete SCO (n = 18). The percentage of SCO tubuli was more strongly correlated to serum inhibin B (r = -0.58; P<0.01) than to FSH (r = 0.34; P<0.05). Similarly, the percentage of tubules with elongated spermatids was significantly (P<0.05) more strongly correlated to serum inhibin B (r = 0.65; P<0.01) than to FSH (r = -0.4; P<0.01). Thus, inhibin B is slightly more sensitive than FSH as an index of the spermatogenic status. Neither FSH nor inhibin B alone, however, could predict the type of spermatogenetic damage exactly. The combination of FSH and inhibin B had high diagnostic sensitivity (88%) and specificity (83%) for the presence of elongated spermatids in testicular biopsies. Sperm could be retrieved in 34 (65%) of the TESE patients. The combination of inhibin B and FSH measurement showed a sensitivity of 75% and a specificity of 73% when identifying patients in whom sperm could possibly be retrieved by TESE. We conclude that although the measurement of serum inhibin B improves the sensitivity of predictive tests for the presence of sperm in histology or for TESE, this parameter cannot accurately predict TESE outcome.

Evaluation of the utilization of dietary nitrogen by dairy cows based on urea concentration in milk.

The objective of this study was to determine whether milk urea concentration is a valuable tool to monitor the utilization of dietary N by dairy cows. Data from 11 feed trials (n = 2828 observations of 356 cows) were used in this study. Dietary protein utilization was evaluated according to the Dutch DVE-OEB system. A close correlation (0.8) was found between rumen-degraded protein balance in the ration and urea concentration in milk. The effects of the balance of true protein digested in the small intestine and net energy on milk urea concentration were small but significant. Parity and stage of lactation did not significantly influence milk urea concentration. Because of the large variation among and within cows, the monitoring of protein utilization of an individual cow was inaccurate. However, milk urea concentration in bulk milk is a valuable tool to monitor the rumendegraded protein balance in the ration.

Estimation of variance components for somatic cell counts to determine thresholds for uninfected quarters.

The objective of this study was to determine the factors affecting somatic cell count (SCC), to estimate variance components of these factors, and to calculate and evaluate the thresholds for intramammary infection based on SCC. The infection status from 22,467 quarter milk samples from 544 cows in seven herds was determined. Infections status was the most important factor affecting SCC. The increase in SCC was more pronounced for major pathogens than for minor pathogens. Even after adjustment for infection status, the interaction between stage of lactation and parity was significant. For culture-negative samples within a lactation, the shape of the SCC curve was inversely related to the shape of the milk production curve. The shape of the SCC curve was flat for first lactation cows compared with the shape of the SCC curve for cows in subsequent lactations. The effect of clinical mastitis on SCC was significant. The use of SCC thresholds for specific parities and stages of lactation to detect intramammary infection improved quality parameters only slightly over a fixed threshold of 200,000 cells/ml.

Activation of oriLyt, the lytic origin of DNA replication of Epstein-Barr virus, by BZLF1.

oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is activated by the viral transactivator BZLF1 which belongs to the extended bZIP class of transcription factors. Seven binding sites for BZLF1, so-called ZRE sites, are located within oriLyt. By mutational analysis of individual ZRE sites, we found that lytic DNA replication is dependent on only four of these sites which colocate with the promoter of the BHLF1 gene. The remaining three ZRE sites distal to the BHLF1 promoter were dispensable for DNA replication and did not contribute to long-range transcriptional activation of this promoter by BZLF1. This finding indicated that a similar set of ZRE sites is involved in DNA replication and transcriptional activation. To determine the function of BZLF1 in DNA replication, BZLF1 mutants with successive deletions in the transactivation domain were analyzed in replication assays. Unexpectedly, most BZLF1 mutants which failed to support DNA replication were found to be equally defective in transcriptional activation. Therefore, similar trans-acting domains of BZLF1 are involved both in replication and in transcription.