The factor time in fetal heart rate monitoring and the detection of acidosis using the WAS score.

BACKGROUND: Using the naked eye evaluation of fetal heart rate (fhr) patterns remains difficult and is not complete. Computer-aided analysis of the fhr offers the opportunity to analyze fhr patterns completely and to detect all changes due to hypoxia and acidosis. It was the goal of this study to analyze the factor time in fetal monitoring and to evaluate the association between the fhr and the actual pH values in arterial umbilical blood. METHODS: During a period of 11 years the FHR signals (i. e., the R-R interval of the F-ECG) of 646 fetuses were recorded with a CTG and simultaneously by a computer. The computer files were analyzed thereafter, i. e., the results did not influence our clinical management. To enter the study, all fetuses must have been delivered by the vaginal route--in consequence without a significant loss of fhr signals. During forceps and/or vacuum deliveries recordings were continued. If necessary a new electrode was inserted. In this study recordings of fetuses with chorioamnionitis, tracings of malformed neonates and tracings shorter than 30 min were excluded. Thus 484 recordings were left. We used our own computer programs written in MATLAB (USA). 3 parameters were determined electronically: 1) the mean fetal frequency [fhf, (bpm)], 2) the number of turning points (N/min) in the fhr, which we called 'microfluctuation' (micro) and 3) the oscillation amplitude, oamp (bpm). Measurements of the acid-base variables from arterial (UA) and venous (UV) blood were performed using RADIOMETER equipment (ABL500) and trained personnel. However, only the actual pHUA values were used in this study. To detect the influence of hypoxia and acidosis, all 484 cases were separated into 7 groups according to the actual pH(UA) value: 55 fetuses lying in a small non-acidotic "pH-window" (pH(UA)=7.290-7.310, mean=7.300±0.008) were used as 'controls'. RESULTS: In humans fhf, micro and the oamp behave differently during the last 30 min of delivery and with different fetal pHUA values: micro is early (at 0 min) decreased with fetal acidemia and is steadily deceasing (68-40 N/min) during vaginal delivery; the oamp--mainly due to decelerations--is increased from 35 up to 70 bpm during the last 30 min. Hypoxia and acidosis increase the amplitude and duration of decelerations; finally fhf shows only an insignificant reaction to acidemia but is decreased (from 135 to 110 bpm) overall with the course of time. Therefore the 3 characteristics of the fhr might be ranged according to their decreasing sensitivity to acidemia as follows: 1) fetal microfluctuation, 2) oscillation amplitude and 3) mean frequency. The 3 components of the fhr were used to invent and apply a score named the WAS score. This score increases the association between the actual pHUA values and the activity of the fetal heart. The 3 variables of the fhr mentioned above were rated differently; the 3 factors necessary to achieve this were computed electronically using an optimization program. The result is the WAS score: WAS=mean [frq*f(f)(v(j)) * micro*f(m)(v(j))/oamp*f(a)(v(j))](j=1,30). Using the last 30 min of delivery the correlation coefficient r of this score with pH(UA) reaches 0.645, P<< 0.001. The regression is linear in our 484 cases. CONCLUSIONS: Microfluctuation is the most sensible variable of the fetal heart followed by the oscillation amplitude and mean frequency. The WAS score offers the best correlation with the actual pH values measured in arterial umbilical blood.

Fetal heart rate patterns during delivery complicated by hypoxia and acidosis - a computer-aided analysis.

BACKGROUND: Using the naked eye, evaluation of fetal heart-rate (FHR) patterns remains difficult and is not complete. Computer-aided analysis of the FHR offers the opportunity to analyse FHR patterns completely and to detect all changes possibly due to hypoxia and acidosis. It was the goal of this study to make these hypoxic changes of the FHR visible and to compare them directly with normal tracings. METHODS: During a period of 11 years the FHR signals (i. e., R-R intervals of the F-ECG) of 646 fetuses were recorded simultaneously also by a computer. The computer files were analysed thereafter, i. e., the obtained results had no immediate influence on the clinical management itself. To enter the study all fetuses must have been delivered by the vaginal route - in consequence without a significant loss of FHR signals. During forceps or vacuum deliveries recordings were continued. If necessary a new electrode was inserted. Recordings of fetuses with chorioamnionitis, tracings of malformed neonates and tracings shorter than 30 min were also excluded. No additional drugs were given to the mother during the time of recording. Thus 484 recordings were left. In this study only the last 30 min of each record were analysed off-line using our own computer programs written in MATLAB. Only 3 parameters were determined electronically: i) the mean fetal frequency (FRQ, bpm), ii) the number of turning points (N/min, see Fig. 1) in the FHR, which we called 'microfluctuation' (MIC) and iii) the oscillation amplitude of the FHR (OA, bpm, Fig. 1). Routine measurements of the acid-base variables from umbilical arterial (UA) and venous (UV) blood were performed using RADIOMETER equipments (ABL500) and trained personnel. To compare acidotic and non-acidotic FHR tracings, 2 pH groups were chosen: fetuses with a small non-acidotic "pH window" (pHUA=7.290-7.310) and 5 fetuses with severe acidosis, i. e., pHUA values <= 7.103. RESULTS: Using this narrow "pH-window" (7.290-7.310) shows that FRQ, MIC and OA belong together. The 3 variables are strongly associated with each other in a linear manner. All 3 correlation coefficients (r) are highly significant (P<0.001); in this context all 3 regressions seem to be linear. The mean pHUA in this special group amounts to 7.300±0.008 (N=50). In severe fetal acidosis (mean pHUA=7.051±0.060, N=5) MIC and OA are diminished significantly (P << 0.001) whereas the mean frequency is increased (ca.+6 bpm). Microfluctuation (MIC) seems to be the most sensible FHR parameter for the diagnosis of hypoxia and acidosis followed by OA and the fetal frequency niveau. CONCLUSIONS: In non-acidotic fetuses MIC, OA and FRQ belong together and their association can be described by the 3 basic principles presented above. Fetal reaction patterns during hypoxia and severe acidosis differ significantly when compared with tracings of non-acidotic fetuses. Computer-analysis reveals that MIC is the most sensitive FHR variable concerning hypoxia and acidosis followed by OA and the mean FRQ niveau. (Some of the acidotic recordings together with the WAS-score can be observed in full length at www.fhr-monitoring.org.).

Basic principles of the foetal heart rate during delivery without hypoxia and acidosis.

BACKGROUND: Using naked-eye evaluation of foetal heart rate (FHR) patterns remains difficult and is not complete. Computer-aided analysis of the FHR offers the opportunity to analyse the FHR completely and to detect all changes due to hypoxia and acidosis. In order to better understand these changes FHR patterns in non-acidotic foetuses should be studied by first separating FHR into (i) basal FHR (baseline) and (ii) all decelerations. METHODS: The FHR signals (i.e., R-R intervals) of 637 fetuses were recorded by a computer. To enter the study all foetuses must have been delivered by the vaginal route - in consequence without a significant loss of FHR signals. During forceps/vacuum delivery recordings were continued. If necessary a new electrode was inserted. Recordings of foetuses with chorioamnionitis and tracings of malformed neonates and tracings shorter than 30 min were excluded. No drugs were given to the mother during the time of recording. Thus 484 recordings were left. In this study only the last 30 min of each record were analysed using our own programmes written in MATLAB. 3 parameters were determined electronically: (i) the mean frequency (FRQ, bpm), (ii) the number of turning points (N/min), which we called 'microfluctuation' (MIC) and (iii) the oscillation amplitude (OA, bpm) (see Fig. 2). Computer analysis of the FHR offers the opportunity to separate baseline FHR from deceleration patterns using appropriate algorithms rearranging and sequencing all baseline segments (or all decelerations) to a new file. Therefore each of the 2 new files contains only one category of the FHR: baseline segments (with accelerations) only or decelerations only (Fig. 1). 1 min was always taken as the reference time interval. In order to exclude foetal hypoxia and acidosis during the last 30 min, a small pHUA -'window' was chosen (7.290 up to 7.310) using acid-base variables from umbilical arterial (UA) blood measured soon after delivery with RADIOMETER equipment (mainly ABL500) by trained personal. RESULTS: Overall 14,520 min of the 484 foetuses were analysed by measuring in UA blood (X ± SD):pH=7.262 ± 0.065, pCO2 = 53.7 ± 8.8 mmHg, BEEcf,ox=-3.3 ± 2.5 mmol/l and sO2 = 23.9 ± 12.4%. In the whole sample and in non-acidotic (pHUA: 7.29-7.31) foetuses (N=50) there exist 3 fundamental principles which combine the 3 FHR variables under investigation: (I) MIC is strongly associated (r=0.631, P << 0.0001) with mean FRQ (bpm): in ca. 40% of all foetal heart beats a turning of the vector occurs (Fig. 4). (II) MIC is associated also with OA (r = -0.480, P << 0.0001); this regression is non-linear: Smaller band-widths are associated with increased MIC [OA = 0.0027 × MIC2 - 0.56 × MIC + 71 (see Fig. 5)]. (III) In non-acidotic foetuses lowering of the mean frequency niveau is associated with increased OA (overall: r = -0.349, P<< 0.0001); Using baseline segments only: r = -0.283, Nmin=844, P<0.0001. This regression is linear again: OABL = -0.445 × FRQBL + 94.1. Overall a Delta frequency (ΔFRQ) of + 10 bpm leads to a ΔOA of -4.1 bpm. These 3 rules are valid in isolated baseline segments as well as during artificially isolated deceleration patterns. CONCLUSIONS: FHR is a unit and should be analysed by computer-aided technologies as a unit. MIC, OA and FRQ belong together and their interaction can be described in non-acidotic foetuses by the 3 basic principles given above. Standard FHR tracings remain untouched.

Sensitivity, specificity, receiver-operating characteristic (ROC) curves and likelihood ratios for electronic foetal heart rate monitoring using new evaluation techniques.

OBJECTIVE: Hypoxia and acidosis adversely influence many foetal organ functions. We wanted to know how foetal heart rate (FHR) patterns are mirrored by the fetal acid-base status and if they could serve for predicting the actual pH in umbilical artery (UA) blood. For this purpose we condensed the FHR phenomena into one figure which was to be used as a testing variable and to analyse the performance of the new testing procedure. METHODS: The direct FHR signals of 475 foetuses were stored in a computer and analysed offline (MATLAB). All foetuses were delivered by the vaginal route thus without a significant loss of signals. The last 30 min of each recording were used. Acid-base variables and blood gases were determined in cord blood (UA and UV) using RADIOMETER equipments. Three variables of the foetal heart rate (FHR) were computed for each minute: oscillation amplitude [oza (bpm)], microfluctuation [ozm (N/min)] and mean frequency [fhm (bpm)]. These variables were combined to a new index, which we call the WAS index: WAS(T)=FHM(T) x OZF(T)/OZA(T). Using optimisation programmes this index was tailored to actual pH, UA leading to the novel, adapted index: WAS(T)=[FHM(T) x GFHM] x [OZF(T) x GOZF] x [OZA(T) x GOZA]-1,where GFHM, GOZF and GOZA denote three mathematical functions comparable to boundaries in discontinuous scoring-procedures, e. g., the APGAR score. The mean of the WAS index for the last 30 min of delivery is called the WAS score and is used as a discriminator in the testing procedure. WAS score and measured pHUA-values were submitted to correlation and linear regression analysis. Sensitivity, specificity, likelihood ratios, and post-test probabilities were computed including their 95% confidence intervals (CI). A ROC analysis was performed by applying different thresholds for pHUA. RESULTS: pH and WAS score are normally distributed in this sample. The correlation coefficient (r) for pHUA and the WAS score amounts to 0.657, P<<10 (-4). Using ROC plots the area under the curve (AUC) is steadily increased with decreasing pHUA reaching 1.0 for pH 7.0 indicating excellent test accuracy. The AUC for pHUA=7.100 is already 0.963+/-0.066, 95% CI (0.942-0.978), P<0.001. The positive likelihood ratios (+LR) far exceed 10.0 when lowering the threshold for pHUA. Aiming at a sensitivity of 100% the discriminatory power of the test becomes clinically an optimum when using a discriminator of 1.816 and a threshold pH of 7.122: Sensitivity=100%, specificity=89.3%, FNR=0%, FPR(%)=10.7% and AUC=0.958+/-0.049, 95% CI (0.936-0.974), P<0.001. CONCLUSIONS: Computer-aided evaluation of FHR patterns leads to a novel index (WAS score) which predicts foetal acidaemia with a high level of accuracy. Therefore online WAS scoring is proposed as an ancillary test procedure for future evaluation of FHR patterns. The conventional EFM remains untouched.

[A new approach to quantitative electronic foetal heart-rate analysis]. / Ein neuer Weg zur quantitativen elektronischen CTG-Analyse.

BACKGROUND: Recently it was found that a significant correlation exists between the variables of the foetal acid-base balance (ABB) and the parameters of the foetal heart rate (FHR). This dependency can be used for diagnostic purposes. Until now FHR could be evaluated off- and online by computer using scoring procedures, i. e., discontinuous methods, which need considerable computational efforts since FHR must reliably be separated into baseline (BL), decelerations and accelerations. Therefore, the question must arise whether a continuous and less cumbersome evaluation of individual FHR-parameters might lead to even better results? METHODS: The last 30 min of 465 direct recordings of foetuses all delivered by the vaginal route were stored in a computer and analysed offline using our own MATLAB programmes. Five variables were computed for every single minute: The microfluctuation, the oscillation amplitude, the mean frequency, the sum of the absolute differences of time periods and the sum of the absolute differences of frequencies. In this paper the last two variables will be abbreviated 'total sums'. All minutes of a FHR tracing were treated equally i. e. there was no separation anymore between BL and decelerations (accelerations). In order to analyse the physiological intercorrelation of the five variables mentioned above a 'pH window' was chosen between 7.275 and 7.325 [7.275< or =pH, (umbilicalartery (UA)< or =7.325]. Thereafter, the influence of hypoxia and acidosis on the five variables was evaluated. According to the results obtained two of the five variables, the 'total sums', were not further analysed. Using the remaining variables 'frequency', 'oscillation amplitude' and 'microfluctuation' a new index, the WAS index, was created. This index offers the opportunity to evaluate the FHR continuously using 'coding lines' for each of the three parameters. The WAS index was designed according to the FHR characteristics of the awake (Wach), acidotic (Azidotisch) (pH, UA=ca. 7,000) and sleeping (Schlafend) foetus. Using the last 30 WAS indices covering the last 30 min of a single FHR tracing, a WAS score and the corresponding prognostic pH value was computed. These prognostic pH values were compared with the measured pH values of UA blood using again correlation analysis. RESULTS: Without any hypoxia and acidosis (pH, UA window) the five FHR variables are closely correlated with each other. Accepting minutes with decelerations only, the correlation between the 'total sums' and the remaining three parameters vanishes. Accepting hypoxia and acidosis (pH, UA > or =6.960) oscillation amplitude now offers the closest correlation with actual pH, UA (r=-0.440 p<<10 (-4)) followed by microfluctuation (r=0.224, p<0.001) and frequency (r=0.056, P: n. s.). This pattern is obviously due to the retention of all decelerations in the FHR tracings. The 'total sums' show less close correlations with pH, UA and BE(oxy) when compared with microfluctuation and oscillation amplitude (SumPER vs. pH, UA r=-0.125, p<0.001 and SumFRQ vs. pH, UA r=-0.154, p<0.001). All five variables under investigation show a better correlation with pH, UA when compared with BE(oxy) UA. The WAS score computed (mean) for the last 30 min of delivery leads to close correlations (p<<10(-4)) with all variables of the foetal ABB: pH, UA r=0.608; BE(oxy) r=0.535, pCO(2) r=-0.469 and sO(2) r=0.259. The median WAS score amounts to 0.176, the mean to 0.174+/-0.023; it is normally distributed in this sample. CONCLUSIONS: FHR characteristics in different foetal behavioural states offer the opportunity to design a new index, the WAS index, which shows close (p<<10(-4)) correlations with all variables of the foetal ABB. Thus, the pH value in cord blood can be predicted within clinically reasonable limits. The qualitative CTG analysis remains untouched.

Fluid regimens in the first week of life may increase risk of patent ductus arteriosus in extremely low birth weight infants.

BACKGROUND: High fluid volumes may increase neonatal morbidity. However, evidence supporting fluid restriction is inconclusive and restricting fluids may restrict caloric intake. OBJECTIVE: To determine if higher fluid intake was associated with increased risk of patent ductus arteriosus (PDA) or bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. STUDY DESIGN: A total of 204 ELBW (170 ml kg(-1) day(-1)) in the first days of life is associated with increased risk of PDA.

[Quantitative Cardiotocography--what does it look like and what can we expect]. / Quantitative Kardiotokographie -- wie sieht sie aus und was dürfen wir von ihr erwarten?

BACKGROUND: [corrected] Cardiotocography (CTG) seems to be a non-reliable, expensive but nevertheless practical method for fetal surveillance. Moreover, its diagnostic value is dependent on a long-standing experience of the obstetrician (midwife). It is difficult to define exact diagnostic criteria since nearly all CTG phenomena are lacking precise qualification by the naked eye. Therefore, the idea must be born in mind to analyze fetal heart frequency (FHF) by computer, first off-line then on-line, in order to evaluate its true diagnostic power objectively. METHODS: The FHF of 583 deliveries terminated by the vaginal route were registered prospectively using a PC and an RS422 interface. In 443 cases acid-base measurements (ABL 500, RADIOMETER, Copenhagen) in blood of the umbilical artery (UA) and vein (UV) were available and plausible. In this study only the last 30 min ante-partum were analyzed. The program for FHF analysis was written in MATLAB (The Mathworks Inc., USA). A CTG score was developed using three components of FHF: basal FHF, the deceleration area of all dips, and the micro-fluctuation (MF) of the basal fetal heart rate (FHR). MF denotes the true number of "turning-points" per minute of basal FHR. For each component a maximum of 6 scoring points could be assigned according to empirical cut-off values. These cut-off values were determined using correlation analysis with acid-base parameters in UA blood, especially the actual pH. The accordance between score and pH values was further demonstrated by assignment of 0.036 pH-units to each of the 19 (18 + zero) scoring points, thus covering a pH range between 6.700 and 7.350 (UA). A resulting variable, delta pH (pH measured, UA-pH assigned) was studied and used for further analysis. In order to define criteria for fetal mortality in utero only cases with pH, UA between 7.250 and 7.350 were accepted. RESULTS: Median basal FHF under normal conditions amounted to 138 bpm (mean: 137 +/- 14.9) in 4180 minutes of 372 fetuses. 120 bpm equals the 13.4(th) and 160 bpm the 94.6(th) centile of the distribution. Given fetal normacidity (UA) MF is 58/minute and the mean MF 57.9 +/- 13.4, respectively, with a 10 (th) centile of 41/minute. MF and basal FHF are correlated significantly (r = 0.410, P << 10(-4)). The declaration-area per fetus is significantly correlated with actual pH (UA), r = -0.473, P << 10(-4). the score itself is highly significantly correlated with actual pH (UA) (r = -0.559, P << 10(-4)) and the other parameters of fetal acid-base balance. Nevertheless, prediction variability for pH, especially in score = 1, 2 and zero (minimal CTG pathology) is still present: 80% of all predicted pH values lie in between -0.092 and + 0.071. It is strongly suggested that this score-related predictive pH variability is caused by maternal breathing habits during the last 30 minutes of delivery. CONCLUSION: Adequate quantification of only three variables of FHR using a score leads to fairly good correlations with parameters of the fetal acid-base balance. Thus actual pH (UA) can be predicted in reasonable clinical limits. Still present variability in prediction of pH seems to be, in part, of maternal origin. The maternal influence could be eliminated by continuous (transcutaneous) monitoring of maternal pCO(2). Along these lines the quantitative electronic monitoring of FHR will be realized and instrumented (off-line and on-line) by nexus/gmt, Frankfurt, a.M., Germany.

Patients with Glanzmann thrombasthenia lacking platelet glycoprotein alpha(IIb)beta(3) (GPIIb/IIIa) and alpha(v)beta(3) receptors are not protected from atherosclerosis.

BACKGROUND: Platelets have been suggested to play a role in the early development of atherosclerosis. As one test of this hypothesis, we assessed whether patients with Glanzmann thrombasthenia who lack platelet glycoprotein alpha(IIb)beta(3) (GPIIb/IIIa) complexes or both alpha(IIb)beta(3) and the more ubiquitous alpha(v)beta(3) cell membrane complexes are protected from development of atherosclerosis. METHODS AND RESULTS: Seven patients with Glanzmann thrombasthenia, 45 to 66 years of age, underwent bilateral carotid artery ultrasonography and screening for risk factors of atherosclerosis. Findings consistent with early atherosclerosis evaluated by measurement of intima-media thickness and presence of atherosclerotic plaques were observed in 6 of the 7 patients. Intima-media thickness values higher than the 75th and 90th percentiles of age- and sex-matched white control subjects of the Atherosclerosis Risk in Communities (ARIC) study were observed in 30 and 8 of 56 carotid artery measurements, respectively. Five of the 6 patients with signs consistent with early atherosclerosis lacked both alpha(IIb)beta(3) and alpha(v)beta(3) complexes and 1 only lacked alpha(IIb)beta(3). CONCLUSIONS: Glanzmann thrombasthenia does not protect affected individuals from development of atherosclerosis.

Treatment of traumatic bleeding with recombinant factor VIIa.

Surgical intervention failed to stop life-threatening bleeding caused by injury complicated by severe coagulopathy. Administration of recombinant factor VIIa immediately corrected the coagulopathy and bleeding stopped.

Mechanism of organic anion transport across the apical membrane of choroid plexus.

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.

Surgical resource utilization in urban terrorist bombing: a computer simulation.

BACKGROUND: The objective of this study was to analyze the utilization of surgical staff and facilities during an urban terrorist bombing incident. METHODS: A discrete-event computer model of the emergency room and related hospital facilities was constructed and implemented, based on cumulated data from 12 urban terrorist bombing incidents in Israel. RESULTS: The simulation predicts that the admitting capacity of the hospital depends primarily on the number of available surgeons and defines an optimal staff profile for surgeons, residents, and trauma nurses. The major bottlenecks in the flow of critical casualties are the shock rooms and the computed tomographic scanner but not the operating rooms. The simulation also defines the number of reinforcement staff needed to treat noncritical casualties and shows that radiology is the major obstacle to the flow of these patients. CONCLUSION: Computer simulation is an important new tool for the optimization of surgical service elements for a multiple-casualty situation.

The alphabet soup of plant intracellular signalling: enter cyclic nucleotides.

Recent work reveals a role for cyclic nucleotides as secondary signalling molecules in a variety of signal transduction pathways in plants. Evidence is accumulating that cGMP is involved in signalling during photomorphogenesis and that cADP-ribose triggers the release of sequestered Ca2+ during the response of plant cells to abscisic acid. Though more tentative, cAMP has been proposed as playing an important role in ion channel activity and cell cycle progression. Taken together, a picture emerges of differing signalling pathways, possibility interacting with each other, acting on an array of developmental processes.

Identification and role of adenylyl cyclase in auxin signalling in higher plants.

Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes, but its significance in higher plants has been generally doubted because they have low adenylyl cyclase activity and barely detectable amounts of cAMP. Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media. The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant. Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe. Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation. Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide. This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

T-DNA tagging reveals a novel cDNA triggering cytokinin- and auxin-independent protoplast division.

Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.

Developmental and auxin-induced expression of the Arabidopsis prha homeobox gene.

A single-copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecular weight of 90,500 has been characterized. The position of the gene was mapped to the distal part of chromosome 4. Expression of the gene differs in various vegetative and floral plant tissues and is positively influenced by the phytohormone auxin. In Arabidopsis plants, a complex pattern of prha promoter-driven GUS expression is observed, often associated with regions of developing vascular tissue. Exogenously applied auxin strongly increased endogenous prha transcript levels. In addition, the prha promoter is highly responsive to the synthetic auxin, naphthalene acetic acid, in transient assays using tobacco protoplasts. The PRHA protein has the capacity to bind to TAATTG core sequence elements but requires additional adjacent bases for high-affinity binding. These findings are discussed in relation to studies of other plant homeobox genes as well as possible in vivo target genes for PRHA.