Biomedical subjects

F Sierra

Publications and source records attributed to F Sierra.

At least 19 recordsLinked to original sources

PubMed2007

Voltage-gated potassium conductances in Gymnotus electrocytes(AB).

Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na(+)-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1-11] there were four K(+)-dependent conductances. Membrane depolarization activated a delayed rectifier (I(K)) and an A-type (I(A)) current. I(A) displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The I(K) activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex "hyperpolarizing responses" (HRs) described under current-clamp. IR1 shows "instantaneous" activation, is blocked by Ba(2+) and Cs(+) and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba(2+) and Cs(+). This current fits the depolarizing phase of the HR. The EOD waveform of Gymnotus carapo is more complex than that of other gymnotiform fish species, the complexity originates in the voltage responses generated through the interactions of three Na(+) and four K(+) voltage- and time-dependent conductances although the innervation pattern also contributes [Trujillo-Cenóz O, Echagüe JA (1989) Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J Comp Physiol A 165:343-351].

Animals↗

PubMed2006

Telemetric monitoring of tracheal pressure after tracheal occlusion for treatment of severe congenital diaphragmatic hernia.

INTRODUCTION: Prenatal tracheal occlusion using endoscopic techniques obstructs the normal egress of lung fluid during pulmonary development and stimulates lung growth in cases of congenital diaphragmatic hernia (CDH). Although FETO might be an effective strategy for treatment of CDH, the mechanism especially due to the supposed increasing transpulmonary pressure is unknown. OBJECTIVE: The purpose of this study was to monitor the pressure below the attached balloon in the fetal lamb telemetrically. METHODS: Four time-dated pregnant Merino ewes underwent fetal and maternal surgery. A special prepared silicone catheter was placed below the epiglottis by laryngoscopy on day 110 or 140 of gestation. The tracheal pressure below the fixed catheter could be monitored telemetrically using the Data Sciences TA11-PA-C40 pressure device. Hundred and twenty measurement points were recorded over a period of 2 min. RESULTS: A maximum of lung pressure rate was found immediately after implantation (23.7 +/- 4.6 mm Hg). During the first hour, the pressure decreased to an average value of 16.9 mmHg. About 70 h after the block, this value decreased to a minimum level of 8.3 +/- 0.4 mmHg. CONCLUSION: Decreasing pressure variation might indicate that lung growth has stopped and that the ideal point of time to remove the balloon is achieved. Increasing pressure has to be related to the morphometric analysis of the lung's structural development and maturation, comparing the efficacy of FETO in preventing or reversing pulmonary hypoplasia. Further investigation of continuous telemetric monitoring of tracheal pressure in the fetal lamb is required.

Animals↗

PubMed2005

T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration.

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).

Aging↗

PubMed2004

Evaluation of the development of lung hypoplasia in the premature lamb.

BACKGROUND: The death rate from human diaphragmatic hernia (CDH) ranges from 50 to 80%, mainly due to the associated lung hypoplasia. To prevent these irreversible pathological and physical defects, the question of intrauterine surgical intervention arises. The histological changes of the lung tissue after inducement of a diaphragmatic hernia were examined. Of special interest was the time elapsing until the development of lung hypoplasia. METHODS: A model of intrauterine inducement of diaphragmatic hernia was established using five fetal lambs to study consecutive pulmonary hypoplasia. Inducement of a diaphragmatic hernia was undertaken between 105 and 108 days' gestation. Lung tissue was examined histologically on postoperative days 8, 17, 21, 22, and 25 after inducement of the defect. RESULTS: On postoperative days 8, 17, and 21, no signs of pulmonary hypoplasia were found on histological examination. A pulmonary hypoplasia was found in two fetuses (on the 22nd and 25th postoperative day). The pathological and anatomical examination of a unilateral pulmonary hypoplasia after a short period of time shows that the artificially created diaphragmatic defect is a good model for producing a congenital diaphragmatic hernia. DISCUSSION: The severity of the pulmonary hypoplasia is related to the duration of lung compression by the herniated organs. The time elapsing until the development of lung hypoplasia is shorter than expected. Tracheal occlusion seems to be an effective strategy for treatment of the defect CDH, but the best technique for achieving occlusion, and particularly the ideal point in time to carry out "Fetendo," are unknown. Further research into this congenital illness is required in order to treat it.

Animals↗

PubMed2004

Continuous telemetric monitoring of fetal oxygen partial pressure during labor.

INTRODUCTION: Upright or ambulatory birth positions are favorable for fetal oxygenation. Studies of fetal oxygenation with regard to maternal position require free maternal mobility. Therefore, telemetry for a fetal sensor for such investigations is a pre-requisite. Telemetry-if technically feasible-could enable monitoring of fetal oxygen partial pressure using an existing sensor without restricting the mobility of the parturient woman. We have developed a telemetry system for use with a fetal transcutaneous partial oxygen pressure sensor (ttcpO(2)) and have studied effects of maternal position and position changes during normal labor. MATERIALS AND METHODS: The monitoring system consists of three parts: the telemetry unit with the ttcpO(2) sensor to transmit the tcpO(2) and the heating output telemetrically, a modified CTG monitor and a personal computer storing the measurements. All data were plotted on the CTG recording paper and fed into a new purpose-designed software, displaying fetal heart rate, the uterine contraction intensity, ttcpO(2) and the heating output. Three laboring women, randomly and successively adopting "classical birth positions" (supine or side positions), sitting or vertical or walking position, were studied. RESULTS: Fetal heart rate, uterine contractions, ttcpO(2) and heating output are influenced by the birth positions and by changes of the birth position. In the classical supine and side position there seemed to be lower fetal oxygenation. Sitting, standing and especially walking were more favorable. DISCUSSION: Telemetry is useful to study a possible clinical benefit of individual birth positions.

Equipment Design↗

PubMed2001

T-kininogen inhibits fibroblast proliferation in the G(1) phase of the cell cycle.

By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblast-derived cell lines that express biologically active T-kininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G(0) cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G(0) through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G(1) and S phases of the cell cycle.

3T3 Cells↗

PubMed2001

[Comparison of sonographic weight estimation to actual birth weight: a retrospective analysis].

AIM: This retrospective study aims at determining the accuracy of sonographic estimation of birth weight based on ultrasound examinations performed at the department of gynaecology of Philipps University at Marburg, Germany. METHOD: 630 children were born at the university department of gynaecology during the first six months of 1998. 519 babies had been examined sonographically and their birth weight estimated within ten days prior to delivery. 176 (33,91 %) of these examinations were carried out by experienced sonographers complying to the level II standard of performance set out by DEGUM. 343 (66.09 %) of examinations were performed by less experienced junior doctors at the time of the mothers' admission to the delivery room. Two reference tables published by Hansmann und Ferrero were used to estimate birth weight. RESULTS: The lower and upper quartile of deviation between estimated weight and actual weight came to -200 g and + 200 g using the Hansmann method and -180 g and + 220 g respectively based on the method of Ferrero. It has to be noted that even greater differences occurred: the 10 % least exact estimates deviated by 500 g and more. The difference between estimated and actual weight increased with the length of pregnancy. The fully trained physician (DEGUM II) tended to estimate the birth weight more accurately. The average birth weight estimated sonographically was lower in "DEGUM II-babies" than that of children whose weight was estimated at the point of admission to the delivery room (Mann-Whitney p < 0,0001). Junior doctors showed a tendency to over-estimate the birth weight of babies actually being underweight. The opposite happened with babies who presented a relatively higher birth weight: their projected birth weight was underestimated. On the other hand, the regression line of the sonographic estimations of birth weight performed by the expert (level II DEGUM) fit to the expected bisector of the angle (Passing-Bablock p > 0,05). CONCLUSION: When clinical decisions are based on estimated values of birth weight, the possible deviation of this value from the actual weight has to be taken into account. In our study this difference came up to 500 g independent of the level of the examiner's ultrasonographic training.

Birth Weight↗

PubMed2000

Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors.

Cell proliferation requires the coordinate synthesis and degradation of many proteins. In addition to the well-characterized involvement of the proteasome in the degradation of several cell cycle-regulated proteins, it has been established that cysteine proteinases are also involved in the control of cell proliferation, but their role is currently not understood. By using both synthetic cysteine proteinase inhibitors and overexpression of T-kininogen (T-KG), a physiologically relevant cysteine proteinase inhibitor, we show that inhibition of cysteine proteinases results in a severe inhibition of the ERK pathway of signal transduction. Mechanistically, this effect appears to be the result of stabilization of the ERK phosphatase MKP-1, which leads to an enhanced dephosphorylation (and hence inactivation) of ERK molecules. These results are specific to cysteine proteinase inhibitors and are not observed when either serine proteinases or the proteasome are inhibited. We hypothesize that inhibition of cysteine proteinases in vivo leads to a dysregulation of the ERK pathway, which results in an inability of the cell to transmit to the nucleus the signals generated by the presence of growth factors, thus resulting in loss of cell proliferation.

Amino Acid Sequence↗

PubMed2000

Impaired signal transduction in mitogen activated rat splenic lymphocytes during aging.

Mitogen activated protein kinases (MAPK) are activated by a wide variety of signals leading to cell proliferation and differentiation in different cell types. With aging, there is a marked decrease in proliferation of T-lymphocytes in response to a variety of mitogens. Several age-related changes in the activation of MAPK pathways in T-lymphocytes activated via the T-cell receptor (TCR) have been described in different species. This way, some TCR proximal defects in tyrosine kinase activity have been delineated. In this study, we have used rat splenic lymphocytes to measure the effect of aging on the activation of two MAP kinase families: ERK and JNK. In order to bypass the receptor-proximal age-dependent defects previously described, we used phorbol ester (PMA) and Ca2+ ionophore (A23187) as co-mitogens. Our results demonstrate that splenic lymphocytes from old rats have a disturbance in the activation of the ERK and JNK MAPK signal transduction pathways, that are located downstream of the receptor-proximal events. At least part of the age-related defect leading to decreased ERK activity appears to be located upstream of ERK itself, since activation of MEK is also impaired. On the other hand, the observed defects in MAPK activation do result in decreased activation of downstream events, such as c-Jun phosphorylation. Thus, we conclude that aging of splenic lymphocytes results in a functional decline in signal transduction, and at least some of these defects are located downstream of the receptor-proximal events previously described by others. The impaired activity of these two MAP kinase pathways is likely to play a role in the diminished lymphoproliferation observed in old individuals.

Aging↗

PubMed2000

Increased expression of mature cathepsin B in aging rat liver.

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.

Aging↗

PubMed2000

Three-dimensional (3-D) ultrasonography for obtaining the four and five-chamber view: comparison with cross-sectional (2-D) fetal sonographic screening.

OBJECTIVES: To assess the ability of Doppler-gated 3-D fetal echocardiography to reconstruct and display specific cardiac structures routinely visualized during antenatal ultrasound in a population at low risk for cardiac anomalies. To determine whether any advantage is offered by 3-D sonographic cardiac examination over conventional sonographic fetal screening techniques. DESIGN: After routine two-dimensional sonographic examination, 3-D cardiac data were collected prospectively in 30 fetuses with gestational ages between 19 and 23 weeks from a low risk patient population. Basic echocardiographic key views were derived from 3-D data and selected for reconstruction and analysis. Four- and five-chamber views were rated and only those views judged to be well visualized were considered as positive results. RESULTS: The four- and five-chamber views were well visualized in all but one fetus using conventional 2-D imaging. Gated 3-D volume data sets enabled visualization of these structures in only 19 of 30 fetuses but provided additional structural depth and allowed a dynamic 3-D perspective of valvar morphology and ventricular wall motion. The right ventricular outflow tract was available from the 3-D volumes in 16 subjects. CONCLUSIONS: Considering the versatility of gated 3-D fetal cardiac imaging we believe that it may soon become an important component of fetal screening thus helping to retrieve standard cardiac cross sections when 2-D imaging is limited by lack of sonographer experience or sonographic windows. Diagnostically acceptable echocardiographic views were obtained more consistently with 2-D ultrasound than with 3-D volume data.

Echocardiography, Doppler↗

PubMed2000

[Prognostic value of Fallopian tube endoscopy].

OBJECTIVE: Progress in sterility-therapy demands diagnostic methods, that ensure the right indication for certain therapies in order to avoid unsuccessful attempts. In this retrospective study we tried to find out, whether we could predict and influence the success rate of therapies with the use of falloposcopy prior to further treatment. MATERIAL AND METHODS: In 62 sterile patients the endotubal state was evaluated with a falloposcope (Imagyn, Irvine, USA), a system of linear everting catheter (LEC) and microendoscope, prior to further treatment. RESULTS: Twenty of the 62 patients had endotubal pathology of both tubes, 15 patients showed pathology of one tube and 25 patients were diagnosed to have a normal endotubal morphology. In 2 patients falloposcopy could not be performed because of blockage due to intramural myomas. Pregnancy rate of those patients with a normal endosalpinx was 52% altogether, following microsurgery 80% and following insemination even 100%. No extrauterine pregnancies were described in this group. Pregnancy rate of those patients with endotubal pathology of both tubes was 35%. In this group no pregnancy followed microsurgery and following insemination one extrauterine pregnancy could be noticed. Furthermore, IVF results were improved if morphologic evaluation showed normal mucosa. CONCLUSIONS: The falloposcopy is a helpful diagnostic procedure to evaluate endotubal pathology prior to microsurgery and insemination.

Adult↗

PubMed2000

Clinical usefulness of pulse oximetry in the fetus with non-reassuring heart rate pattern?

UNLABELLED: The objective of this study was the evaluation of intrapartum pulse oximetry as an indicator of fetal distress and the condition of the newborn during clinical routine surveillance in an University Perinatal Center. Between 1998 and 1999 pulse oximetry (SpO2) was used additionally to routine fetal monitoring by electronic fetal heart rate tracing (CTG) and fetal blood sampling (FBA) in 128 cases with nonreassuring heart rate pattern. Cut off values were FIGO Score < 8 for the heart rate pattern and for fetal blood sampling during labor results of < 7.25 (preacidosis). The condition of the newborn was defined by the APGAR score with the cut off < 7 at 1 minute, while the biochemical status was evaluated by means of arterial blood sampling of the umbilical artery directly after birth using a pH of < 7.20 to verify acidosis. Predictive values of critically low SpO2 values (< 30%) for at least 10 minutes as well as corresponding sensitivities and specificities were calculated together with 95% confidence intervals to identify fetal distress or a depressed condition of the newborns. Of 128 fetuses included in this study 66 (52%) were born spontaneously, 23 (18%) were born by operative vaginal delivery and 39 (31%) by means of cesarean section. The high rate of cesarean section was due to cephalopelvic disproportion in 29 cases. Fetal outcome was evaluated with a clinical score: mean APGAR score value 8.5 SD +/- 1. The mean value of the pH in the umbilical artery was 7.23 +/- 0.04. During a SpO2 monitoring period of 18,381 minutes we analyzed a contact time of 63%. Comparing SpO2 values of < 30% with preacidosis in the fetal blood sampling, we found a positive predictive value of merely 0.17 (95% CI: 0.00-0.64). Of 9 preacidotic cases during delivery only 1 was indicated by a saturation value below 30% (sensitivity 0.11, 95% CI: 0.00-0.48). The specificity and negative predictive value were calculated as 0.83 (95% CI: 0.65-0.94) and 0.76 (95% CI: 0.58-0.89) respectively. Of eleven cases with acidosis in the blood of the umbilical cord artery, pH < 7.20, only 2 were indicated by a SpO2 values below 30%. Which is equivalent to a sensitivity of 0.18 (95% CI: 0.03-0.52). Results of a receiver operator curve analysis showed no substantial deviation from the diagonal. The area under the curve was 0.62, the 95% CI (0.47-0.76) indicating no significant discrimination. Three of 49 fetuses with SpO2 recording during the last 10 minutes were born in clinical depressed status (APGAR < 7). None was indicated by a SpO2 value below 30%. CONCLUSION: Fetal distress and impaired condition of the newborn are not identified or predicted during routine application of SpO2 monitoring in the fetus during labor with adequate safety.

Acidosis↗

PubMed1998

T-kininogen is a biomarker of senescence in rats.

We have previously reported on the identification of T-kininogen (T-KG) as a gene whose expression is increased during senescence in male Sprague-Dawley (S-D) rats. Serum T-KG levels increase 2.5-4 months before the time of death for any given animal, irrespective of the actual age of the animal at the time of this event. Furthermore, dietary restriction (DR) delays, but does not prevent, the increase in serum T-KG levels. In the present study, we have assessed whether or not the age-related increase in T-KG is a common feature of senescence in other strains of rat. We have analyzed hepatic T-KG mRNA levels in male Fischer 344 rats (F344), as well as in male and female (Fischer 344 x Brown Norway)F1 rats (F1). In both of these strains, we observed a dramatic increase in hepatic T-KG mRNA levels when male rats approach senescence. The mRNA levels behave similarly in F1 and S-D rats, in that the increase occurs late in life, and it is either repressed or delayed by DR. In contrast, the increase in T-KG mRNA levels in F344 rats occurs earlier in life, and is not significantly affected by DR. Young female F1 rats fed ad libitum (AL) show a statistically significant (P = 0.0009) 2.6-fold higher level of T-KG mRNA, as compared to their male counterparts. Thus, while we still observe an age-related increase in this parameter in both AL and DR female F1 rats, the difference is statistically significant (P = 0.0001) only in DR animals. We conclude that the increase in T-KG gene expression is a common feature of senescence and that, at least in males of these commonly used rat strains, T-KG can be used as a reliable biomarker of aging. Since the increase in T-KG gene expression does not appear to correlate with inflammatory processes, and since different strains of animals succumb to different pathologies, these results further suggest that the increase in T-KG expression might be related to the process of aging per se, rather than to any given age-related pathology.

Aging↗

PubMed1998

Changes in hepatic DNA binding proteins as a function of age in rats.

The process of aging is accompanied by many changes in gene expression, occurring in virtually all organs of the affected individual. Here we report on the relative changes in DNA binding activity of a panel of 15 different transcription factors in the liver of adult (15-month-old) and old (25-month-old) Sprague-Dawley rats. When expressed as a function of nuclear protein concentration, a great majority of the transcription factors analyzed do not show significant differences in DNA binding activities as a function of age, except activator protein 1 (AP-1) and nuclear factor-kappa B, both of which show increased activities in the older animals, and hepatocyte nuclear factor-3, which undergoes a switch from predominantly alpha and beta subspecies in the adults, to predominantly gamma subspecies in the old animals. Further examination of some of the members of the AP-1 complex using Western blot analysis indicates that the increase in binding activity of this particular complex might be due to an increase in the relative mass of Jun B, presumably resulting in a switch from predominantly c-Fos/Jun D in the young to c-Fos/Jun B complexes in the old animals. Nuclear extracts prepared from the liver of old animals yield less proteins per mass of DNA than similar extracts prepared from younger animals. Accordingly, if the data are analyzed as a function of genomic DNA, our results indicate that aging results in a consistent, but generally not statistically significant decrease in most transcription factor DNA binding activities, with AP-1, nuclear factor-kappa B, and transcription factor II D being the exception to this decline.

Aging↗

PubMed1997

Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins.

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.

Animals↗

PubMed1997

Cloning of the human twist gene: its expression is retained in adult mesodermally-derived tissues.

We have previously reported on the isolation of several differentially expressed genes derived from young and senescent non growing WI-38 human fetal lung-derived fibroblasts. A 0.8-kb cDNA clone, isolate EPC-A2 (early population doubling cDNA-A2), encodes the 3' end of the human homolog of the Twist protein. Twist genes encode basic helix-loop-helix DNA-binding transcription factors that play crucial roles in mesoderm development. Here, we report the cloning and sequencing of the genomic human twist gene. It encodes a protein of 201 amino acids with 96% amino acid sequence identity to mouse Twist, and 100% sequence conservation in the DNA-binding region among all species in which it has been characterized. We further show that expression of human twist is retained in mesodermally-derived tissues and cell lines derived from adult donors.

Adult↗

PubMed1997

Different voltage-gated sodium currents are expressed by human neuroblastoma NB69 cells when cultured in defined serum-free and in astroglial-conditioned media.

Voltage-gated Na+ currents (INa) were analysed with the whole-cell patch-clamp technique in human neuroblastoma NB69 cells plated in serum-free "defined" medium (DM) or in "astroglial-conditioned" medium (CM). Cells survived in both media and expressed the microtubule associated protein 1A, indicating neuron-like differentiation. Two INa types with different time-, voltage-dependent properties and tetrodotoxin (TTX) sensitivities were expressed in DM and CM. The INa in DM-plated cells was present from day 4 and its surface density increased from 11 pA/pF (days 5-7) to 68 pA/pF (days 15-30). The underlying conductance (GNa) half-activated (V0A) at -24 mV. INa inactivation was fitted by single exponentials with 7.5 ms time constant (th) at the -35 mV half-inactivation voltage (V0I). INa was not affected by 10 nM, was reduced (65%) by 100 nM, and not completely abolished (92%) by 300 nM tetrodotoxin (TTX). The INa of CM-plated cells appeared at day 3-4 and its surface density increased from 14 pA/pF (days 3-6) to 28 pA/pF (days 11-14). The GNa V0A was -29 mV and inactivation was fitted by single exponentials with 2.6 ms that the -58 mV V0I. This INa was reduced (55%) by 10 nM and totally abolished by 100 nM tetrodotoxin (TTX). In conclusion, NB69 cells displayed a slow, "TTX-resistant," or a fast, "TTX-sensitive" INa in DM and CM, respectively, suggesting that the CM contained diffusible trophic factors of astroglial origin that induced the expression of a different Na+ channel type. About half of the CM- and DM-plated cells also displayed a persistent Na+ current (INaP).

Action Potentials↗