A role of the tuberous sclerosis gene-2 product during neuronal differentiation.

Tuberous sclerosis is an autosomal dominant disorder. Besides the development of benign growths (hamartomas) in different tissues, one hallmark of this disease is the presence of highly epileptogenic dysplastic lesions in the cerebral cortex (tubers) composed of abnormal shaped neurones. Patients often show evidence of severe mental retardation. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid putative tumour suppressor protein, tuberin, that functions as a GTPase-activating protein. Here we show that tuberin expression is upregulated upon induction of neuronal differentiation in the neuroblastoma cell lines SK-N-SH and LAN-1. This upregulation occurs at post-transcriptional level and is independent of the proliferation status. TSC2 expression is unaffected during differentiation of C2C12 myoblasts into myotubes and of F9 embryonal carcinoma cells into cells resembling parietal endoderm. Antisense inhibition of tuberin expression in SK-N-SH or LAN-1 cells inhibits neuronal differentiation, but does not affect the differentiation of F9 cells. Ectopic overexpression of TSC2 not only reverts the antisense-associated phenotype but furthermore accelerates the neuronal differentiation process. Our data show for the first time that tuberin plays a critical role in neuronal differentiation. Such role is consistent with the phenotype of tuberous sclerosis patients, who inherit one defective TSC2 allele, and frequently lose the remaining normal allele in many of the tubers/hamartomas which develop in the central nervous system of these patients.

Activation of c-Myc uncouples DNA replication from activation of G1-cyclin-dependent kinases.

Proto-oncogenes like c-myc are thought to control exit from the cell cycle rather than progression through the cell cycle itself. We now present a different view of Myc function. Exponentially growing Rat1-MycER fibroblasts were size-fractionated by centrifugal elutriation. In these cells, activation of cyclin E- and cyclin A-dependent kinases, degradation of p27, hyperphosphorylation of retinoblastoma protein and activation of E2F occur sequentially at specific cell sizes. Upon activation of Myc, however, these transitions all occur simultaneously in small cells immediately after exit from mitosis. In contrast, Myc has no discernible effect on the cell size at which DNA replication is initiated. These data show first that Myc controls the activity of G1 cyclin-dependent kinases independently from the transition between quiescence and proliferation and from any effect on cell growth in size. These data also provide evidence of at least one dominant mechanism besides activation of E2F and of cyclin E/cdk2 kinase, which prevents DNA replication unless a critical cell size has been reached.

Fetal cells in the peripheral blood of pregnant women express thymidine kinase: a new marker for detection.

Fetal cells occur in maternal blood in a substantial proportion of normal pregnancies. Several different approaches have been used to detect and enrich these cells for non-invasive prenatal diagnosis. However, before these fetal cells can routinely be used for prenatal diagnosis, perfectly reproducible procedures for detection and enrichment need to be established. We found that these fetal cells express high intracellular levels of the DNA precursor pathway enzyme thymidine kinase. Since normal adult peripheral blood cells do not exhibit any thymidine kinase activity, this enzyme is a potent new marker to detect and enrich fetal cells from maternal blood. We further describe the first successful application of a cytofluorometric thymidine kinase assay to detect fetal cells in the maternal circulation by virtue of their high thymidine kinase activity.

Expression of the cyclin-dependent kinase inhibitor p16 during the ongoing cell cycle.

It has been demonstrated that protein expression of p16, the inhibitor of cyclin-dependent kinase 4 and 6, increases 4 fold at the G1/S transition when serum-arrested cells are restimulated to logarithmic growth. We examined the cell cycle regulation of this cyclin-dependent kinase inhibitor in cells separated according to their cell cycle phases by centrifugal elutriation. Neither p16 mRNA nor its protein expression are regulated during the cell cycle of normal phytohemagglutinin-stimulated lymphocytes, retinoblastoma protein-negative cells, papilloma virus-transformed cells, and acute promyelocytic leukemia cells. p16 mRNA is constitutively expressed in cells in which we detected the normal E2F-dependent S-phase specific expression of thymidine kinase mRNA. We further observed a G1-phase specific expression of cyclin D1 mRNA in the same cells separated by centrifugal elutriation.

Specific transformation abolishes cyclin D1 fluctuation throughout the cell cycle.

We analysed cyclin D1 mRNA and protein expression in several different cell types after separating these cells according to their different cell cycle phases by centrifugal elutriation. In normal human and rat fibroblasts cyclin D1 expression is high in early to mid G1 and decreases about 6-7 fold before onset of replication. It has been demonstrated that specific transforming events, such as loss of functional retinoblastoma protein, overexpression of c-myc, and transfection with the human papillomavirus oncoproteins E6 and E7 cause transcriptional downregulation of cyclin D1 expression in logarithmically growing cells. We found that such transformed cells exhibit loss of the cell cycle-dependent cyclin D1 fluctuation accompanied with reduced upregulation of cyclin D1 in G1 phase. The data presented here provide the experimental support for a recently suggested model involving the function of the retinoblastoma protein in cyclin D1 cell cycle regulation.

Cellular targets for activation by c-Myc include the DNA metabolism enzyme thymidine kinase.

Although a remarkable number of genes has been identified that are either activated or repressed via c-Myc, only few of them obviously contribute to Myc's biological effect--the induction of proliferation. We found that in logarithmically growing cells overexpression of Myc specifically induces thymidine kinase (TK) mRNA expression and enzyme activity, whereas loss of one allele of Myc causes downregulation of this enzyme. We show that activation of Myc triggers high levels of this normally strictly S-phase-regulated DNA metabolism enzyme in serum arrested G0 cells and causes high and constant levels of TK expression throughout the entire ongoing cell cycle. Induction of TK by Myc requires an intact transcriptional activation domain. Myc-induced deregulation of this enzyme is paralleled by alterations of protein binding at the E2F-site of the TK promoter. We further show that cell growth arrest by the cyclin-dependent kinase inhibitor p16 is abrogated by overexpression of Myc and that co-overexpression of p16 cannot inhibit the Myc-induced up-regulation of TK expression. Our data demonstrate TK to be a cellular target of Myc independently of the status of cell proliferation and provide evidence that the transcription factor E2F might be involved in this process.

Loss of the p16/MTS1 tumor suppressor gene causes E2F-mediated deregulation of essential enzymes of the DNA precursor metabolism.

Homozygous deletions of the tumor suppressor gene p16/MTS1 were reported in a wide variety of tumors and tumor cell lines. Its product inhibits the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and CDK6. Because phosphorylation of pRb is a major regulatory event in the activation of the transcription factor E2F, a role for p16 in the regulation of E2F-dependent transcription was presumed. We investigated the effect of the loss of p16 on E2F-mediated transcription in a tumor progression model consisting of three cell lines originating from a common precursor cell--one p16-positive cell line established from the primary biopsy and two lines derived from more advanced stages of the tumor representing the same cell clone after loss of p16. We observed up- and deregulation of E2F-dependent transcription during the cell cycle of the p16-negative cell clones, which returned to normal after transient expression of p16. This p16-dependent regulation affects a set of enzymes necessary for the activation of all four DNA precursors; it is paralleled by the interconversion of transcriptionally active free E2F and transcriptionally inactive higher molecular complexes of E2F and is dependent on the existence of endogenous pRb. Furthermore, we show that p16-negative cell clones exhibit a growth advantage compared to their p16-positive counterparts. One might speculate that one feature of tumor progression could be deregulation of E2F-dependent transcription caused by loss of p16.

Cystosonography: a diagnostic adjunct for the staging of advanced gynecologic malignancies.

Thirty-five patients with advanced gynecologic malignancies (cervical cancer, N = 26; endometrial cancer, N = 5; ovarian cancer, N = 2; vaginal cancer, N = 2) underwent cystoscopy and subsequent cystosonography. The mean age was 61.6 years (range 33-78). The bladder wall was found intact in three patients regardless of the diagnostic procedure. A mere displacement of the bladder wall was observed in four cases using cystoscopy. In all of these women, tumor invasion was ascertained by cystosonography alone. Edema of the bladder mucosa was found in 14 women by cystoscopy and in six women by cystosonography. Endosonography detected tumor invasion of the external layers of the bladder in eight of 14 women. In six of 14 patients, edema was caused by bladder displacement without neoplastic infiltration. Tumor invasion of the bladder was ascertained in six women by cystoscopy and intravesical sonography. Both cystoscopy and intravesical sonography demonstrated neoplastic infiltration and edema of the bladder mucosa in eight patients. Compared with cystoscopy, cystosonographic demonstration of the perivesical region provided better evaluation of the extent of bladder invasion. Cystosonography is a diagnostic adjunct to mere cystoscopy for patients with gynecologic malignancies. This modality is an excellent diagnostic tool, especially if cystoscopy reveals pathologic findings such as edema and displacement or invasion of the bladder wall.

A new method for sonographic urethrocystography and simultaneous pressure-flow measurements.

By the use of a vaginal scanner and simultaneous pressure-flow measurements, we developed a new method to study bladder and urethral function. During urodynamic pressure measurements, a vaginal scanner was positioned adjacent to the vulva just underneath the external urethral orifice to scan the bladder, the vesicourethral junction, and the urethra. This technique was termed "introital sonography." Twenty patients with the symptom of "loss of urine" and ten healthy volunteers without urinary symptoms were studied. Introital sonography displayed the bladder, the urethra, and the symphysis in all women. No incontinence was demonstrable in the healthy control group. Opening of the bladder neck was always visible during micturition, and a normal flow pattern could be observed in all healthy women. In five patients with detrusor instability, opening of the bladder neck was found during cystometry, displaying isolated detrusor contractions. Fifteen patients with genuine stress incontinence demonstrated marked descent of the vesicourethral junction under stressful situations. For the diagnosis of detrusor instability, sonographic visualization of bladder neck opening confirms the entity of isolated detrusor contractions during cystometry and thus helps to exclude tonometric artifacts. Introital sonography not only confirms the diagnosis of genuine stress incontinence by accurately demonstrating the abnormal vesicourethral anatomy, but also provides information that is essential for selecting the proper operative procedure. The lack of radiation exposure and contrast medium makes this inexpensive technique most useful for long-term video pressure-flow studies in patients with bladder dysfunction.

Endosonography in obstetrics and gynecology: the importance of standardized image display.

The use of endosonographic methods (transvaginal sonography, transrectal sonography, cystosonography, hysterosonography) is increasing for diagnostic and therapeutic procedures in obstetrics and gynecology. Because different means of description lead to confusion, endosonography needs a defined method of orientation. The topography used for transabdominal sonography should also be employed for endosonography. When performing transabdominal sonography, the scanner surface is always projected to the top of the screen, regardless of whether the patient is investigated from a ventral or dorsal position. With endosonography, the contact surface of all linear arrays, curved arrays, and sector scanners should be projected to the bottom of the screen. Using a frontally radiating sector scanner or curved array for a sagittal section, the left side of the screen should correspond with dorsal and the right side with ventral; for all other sections, left and right must be defined. A standardized way of displaying images obtained by endosonography should project the scanner surface to the bottom of the image and can be used on all available technical equipment without major difficulty. The advantage of displaying endosonographic images in this standardized manner is the simple differentiation of pictures obtained by an endosonographic or transabdominal technique.

Umbilical artery Doppler velocimetry in fetuses with a single umbilical artery.

OBJECTIVE: Doppler waveform analysis of the umbilical artery is an important tool for the evaluation of high-risk pregnancies. Yet, available data are based on normal values from three-vessel umbilical cords. Our purpose was to evaluate the value of umbilical artery Doppler velocimetry in fetuses with a single umbilical artery. METHODS: One hundred thirteen consecutive singleton fetuses with a single umbilical artery between 16 and 40 weeks' gestational age were studied prospectively at a tertiary referral center for prenatal diagnosis and therapy. Complete follow-up was obtained from 103 cases. RESULTS: The systolic-diastolic ratio in the umbilical artery was abnormal in 31 fetuses (30%) and normal in 72 fetuses (70%). Fetuses with abnormal Doppler waveform analysis in the umbilical artery were significantly more likely to be growth restricted (55 compared with 15%), to have complex malformations (58 compared with 1%) or an abnormal karyotype (29 compared with 0%), or not to survive the fetal/perinatal period (42 compared with 0%) than those with normal Doppler waveform analysis. CONCLUSION: Fetuses with a single umbilical artery and abnormal umbilical Doppler velocimetry had a significantly increased risk of adverse fetal and neonatal outcome compared with those with a single umbilical artery but normal Doppler studies.

Vaginosonography for recording of cycle-related myometrial contractions.

It has been recognized from experimental or invasive studies that the nonpregnant human uterus has an inherent contractibility. We used vaginosonography for imaging contractions of the inner third of the myometrium. The direction, frequency, and symmetry of contractions were noted. We studied 53 women and subdivided them into four groups based on the cycle phase. During menstruation we found contractions toward the cervix with irregular frequency varying between 1 and 3/min. In the periovulatory period we noted the highest frequency of 10/min of regular contractions toward the fundus. The results showed that active myometrial contractions can be detected sonographically throughout the whole menstrual cycle. Increased myometrial contractions toward the fundus in the periovulatory period may be involved in sperm transport to the tubes.

Transvaginal pulsed Doppler measurement of blood flow velocity in the ovarian arteries during cycle stimulation and after follicle puncture.

In experimental studies, an increase of the ovarian blood flow was found during cycle stimulation. In this study, the authors performed transvaginal pulsed Doppler measurements of the ovarian arteries in stimulated cycles before and after follicle puncture. Four days before follicle puncture, high flow velocities in systolis were found compared with diastolis. Toward the day of embryo transfer, a marked increase of the diastolic blood flow velocity was observed. In patients with high endocrine response, the pulsatility index (PI) was significantly lower compared with that of patients with low endocrine response. The technique of transvaginal pulsed Doppler measurements offers the possibility to study the alterations of the ovarian blood flow under physiologic and pathophysiologic conditions.

Endometrial volume change during spontaneous menstrual cycles: volumetry by transvaginal three-dimensional ultrasound.

OBJECTIVE: At present, only limited data are available on endometrial volume during the menstrual cycle. Most of these studies deal with animal models and use magnetic resonance imaging for volume measuring. The application of three-dimensional ultrasound in endometrial volume estimation is the subject of this study. SETTING: Patients visiting the outpatient unit of the division of endocrinology and reproductive medicine of a university hospital. PATIENT(S): Twenty patients with a history of a normal menstrual cycle were selected. INTERVENTION(S): Ultrasound examinations were performed during a single menstrual cycle in addition to routine laboratory tests. MAIN OUTCOME MEASURE(S): Uterus-endometrial volume ratio. RESULT(S): Data from 18 patients could be evaluated. In 81 examinations the endometrium volume could be determined. Mean endometrial volume measured by three-dimensional ultrasound was 1.23 cm3. Mean uterus volume was 48.93 cm3. The change of the uterus-endometrial volume ratio showed a good correlation with the day of menstrual cycle. Quadratic regression analysis of volume and cycle length was R2 = 0.432. CONCLUSION(S): Three-dimensional ultrasound allows assessment of volume data of the female internal genitalia. In this study changes of the endometrial volume in menstrual cycles were measured. Additional studies are required to give information on the clinical impact of this new technique of endometrial volume estimation.

Stem cell marker expression in human trisomy 21 amniotic fluid cells and trophoblasts.

Down Syndrome is the most frequent genetic cause of mental retardation. Deregulation of specific differentiation processes is a major cause for the neuropathological cell features typical for this syndrome. The molecular mechanisms leading to Down Syndrome are likely to be operative from the very earliest time of embryonic/fetal development. We therefore analysed human amniotic fluid cell samples and cytotrophoblastic cells from placental biopsies, both with normal karyotypes and with trisomy 21, for the mRNA expression of stem cell marker genes. Here we describe for the first time that these human primary cell sources contain cells that express telomerase reverse transcriptase, leukemia inhibitory factor receptor, and bone morphogenetic protein receptor II. A specific difference between aneuploid and normal cells could not be detected. These data provide evidence that human amniotic fluid and cytotrophoblastic cell cultures might provide a new source for research on primary cell systems expressing these stem cell markers. In addition, it is suggested that early deregulation of the expression of these genes in the here analysed cell sources does not contribute to the molecular development of Down Syndrome.

Estimation of fetal age by echographic diagnosis of ossification centres in the fetal knee.

The accuracy of term--prediction by echographic demonstration of the upper tibial and the lower femoral centre in patients with unknown confinement dates has been compared. The best prediction was given in case of the presence of tibial epiphysis.

Vaginosonographic Doppler velocimetry in both uterine arteries: elevated left-right differences and relationship to fetal haemodynamics and outcome.

Blood flow velocity was measured in both main stem uterine arteries by means of a transvaginally inserted Duplex scanner (240 degrees sector, pulsed Doppler) and visual vessel recognition to investigate normal and abnormal uterine perfusion. Its relationship to fetal circulation and fetal outcome was studied. Fetal vessels were investigated transabdominally. One hundred and seventy-six pregnancies with a high-risk for fetal malnutrition were examined between the 27th and the 40th week of gestation. In 113 (64%) patients we found normal uterine perfusion (A/B ratios in both uterine arteries less than 3, left-right difference less than 1) and in 63 (36%) cases the A/B ratios were outside our limits. A single abnormal A/B ratio in one of the uterine arteries or an abnormal left-right difference was classified as a mild form of abnormal uterine perfusion. Involvement of both uterine arteries was classified as a severe form. The severe form was associated with a higher frequency of pathological waveforms in fetal arteries and reduced fetal outcome. Clinically, velocimetry in both uterine arteries is of paramount importance when the degree of abnormal uterine perfusion is to be classified precisely.

Three-dimensional ultrasonographic imaging of fetal tooth buds for characterization of facial clefts.

The purpose of this prospective study was to investigate whether the antenatal characterization of fetal facial clefts can be improved by three-dimensional ultrasonographic visualization of fetal tooth buds. Between January 1996 and June 1998, seventeen consecutive fetuses with facial clefts were examined for fetal maxillary tooth buds in the cleft area using three-dimensional multiplanar reconstruction. It was possible in all cases to classify the clefts either as cleft lip alone or unilateral cleft lip and palate or bilateral cleft lip and palate. Three-dimensional computed tomography and histological jaw sections of three stillborn infants were produced in order to examine the correlation between the sonographic, radiographical and histological findings. The prenatal characterization of the facial clefts by means of a visualization of the tooth buds showed to be accurate postnatally in all cases. The sonographic proof of tooth buds might gain increasing importance as this technique seems to facilitate and improve the prenatal classification of suspected facial clefts.

Association of fetal and maternal carboxyhemoglobin levels in normal and Rh-alloimmune pregnancies.

OBJECTIVE: To compare paired antepartum fetal/maternal COHb ratios in whole blood from control and alloimmunized pregnancies and to examine the relationships between fetal and maternal COHb. METHODS: COHb levels were measured in paired fetal and maternal blood samples obtained at cordocentesis in 47 control and 16 Rh-alloimmunized pregnancies. COHb was determined by gas chromatography. Results were analyzed by t-test, regression and analysis of covariance. RESULTS: Although fetal/maternal COHb ratios for control and alloimmunized pregnancies were not statistically significantly different, i.e. 1. 11+/-0.04 and 1.26+/-0.09, respectively (P=0.09), fetal COHb levels were higher in Rh-alloimmunized fetuses (P=0.0002). Fetal COHb levels were also higher than paired maternal levels among the alloimmunized group (P=0.011), but not among the control group (1. 04+/-0.04, P=ns). In univariate regression analysis, fetal and maternal COHb levels were significantly correlated with one another in both control (r=0.52, P=0.0002) and alloimmunized pregnancy groups (r=0.52, P=0.05). Comparison of the slopes of the fetal versus maternal COHb plots for the two groups showed a significant difference (P=0.02), with the alloimmunized group having the steeper slope. CONCLUSION: Differences in the antepartum fetal-maternal COHb relationships in control and alloimmunized groups likely reflect increased endogenous CO production among alloimmunized fetuses as a result of pathologic hemolysis.

Follicular aspiration for in vitro fertilization: sonographically guided transvaginal versus laparoscopic approach.

Laparoscopy is the most commonly used procedure for oocyte retrieval in an in-vitro fertilization (IVF) program. In this study we compared the results of 21 laparoscopic and 21 sonographically guided transvaginal oocyte retrievals using a vaginal probe. Laparoscopically 3.7 and transvaginally 4.8 oocytes per patient were recovered. Overall 6 pregnancies were achieved, giving a pregnancy rate of 14.1% per patient. Vaginal follicular aspiration resulted in a higher oocyte recovery rate. The advantages of the method were a shorter operation time, a superficial anesthesia and, compared to laparoscopy, a less invasive and simpler technique. Therefore this method is now commonly used for routine IVF procedures in our institution.