Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells.

Akt, when activated by IGF/insulin, can phosphorylate forkhead transcription factors. We undertook this study to determine whether epidermal growth factor (EGF) treatment could produce a signaling cascade resulting in phosphorylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231. After establishing ErbB1, cbl, PI3 kinase and Akt were activated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR antiserum that the electrophoretic mobility of FKHR was retarded after EGF treatment. This mobility retardation was reversible by treatment with alkaline phosphatase, and immunoblot with phospho-Ser256 FKHR antibody further confirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY294002, and the ErbB1 inhibitor AG1478. FKHR immunoblotting after purification of nuclear and cytoplasmic proteins showed that EGF induced a simultaneous increase of FKHR in the cytoplasm and decrease in the nucleus. This finding was confirmed by immunofluorescence staining. Treatment of cells with pharmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKHR after EGF treatment by a PI3 kinase dependent mechanism, and represent the first report of growth factor regulation of endogenous FKHR localization.

Aberrant regulation of transforming growth factor-alpha during the establishment of growth arrest and quiescence of growth factor independent cells.

Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.

Regulation of transforming growth factor alpha expression in a growth factor-independent cell line.

Aberrant transcriptional regulation of transforming growth factor alpha (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype. In this paper, we report on TGF alpha promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGF alpha and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGF alpha autocrine activation is the major stimulator of TGF alpha expression in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to the TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGF alpha stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGF alpha-dependent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGF alpha promoter activity in the growth factor-independent phenotype.

Coagulation protein function. IV. Effect of acetaldehyde upon factor X and factor Xa, the proteins at the gateway to the common coagulation pathway.

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russell's viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohol's primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Coagulation protein function. III. Effect of acetaldehyde upon the activation of prothrombin.

When prothrombin is mixed with acetaldehyde (AcH), the primary metabolite of ethanol, at room temperature for 30 min, dialyzed to remove excess AcH, and then activated with Echis carinatus venom (ECV), a prolonged clotting time is observed. Similarly treated prothrombin, however, readily hydrolyzes the synthetic substrate, benzoyl-DL-arginine-beta-naphthylamide (BANA). These results suggest that AcH does not react with the catalytic site of thrombin, which is protected in the prothrombin molecule. However, it does react with susceptible sites on the prothrombin surface which remain alkylated during extensive dialysis to remove excess AcH and subsequent activation of the molecule by ECV. These alkylated sites on the newly formed thrombin molecule may inhibit or prevent the effective/efficient binding of fibrinogen at its binding sites, causing prolonged clotting times. The binding sites for accommodating fibrinogen on the thrombin molecule are apparently quite different from those that bind BANA. These data further suggest that prolonged clotting times that are reported in chronic alcoholics may be due, in part, to ready interaction of acetaldehyde with circulating prothrombin. Fibrinogen, which has been exposed to AcH and subsequently dialyzed to remove excess AcH, also responds with a prolonged clotting time in the presence of added thrombin, signifying that sites on fibrinogen that are essential for its function/conformation are susceptible to AcH.

Ventilatory and timing parameters in normal horses at rest up to age one year.

The purpose of the study was to document the developmental changes in the ventilatory and timing parameters associated with quiet breathing at rest in awake, standing horses during the first year post partum. Tidal volume (VT), breathing frequency, airflow, mechanical timing intervals and minute ventilation (VE) were measured serially in foals age 24 h-1 year. In the growing foal, VE increased due to a progressive rise in VT, in spite of a pronounced decrease in respiratory frequency. When normalised to body weight (bwt), VE/kg declined with maturation in a curvilinear fashion, from mean +/- s.d. 848 +/- 231 ml/min/kg in the 24 h-old foal, to 155 +/- 15 ml/kg/min in the 1-year-old foal. Tidal volume normalised to bwt remained relatively constant during the study period, with the exception that at age 3 weeks and from 2-6 months, VT/kg was significantly lower than the value recorded at age 1 week. The decrease in frequency resulted from prolongation of both inspiratory (TI) and expiratory (TE) time but there was a disproportionately larger increase in TE compared to TI, which resulted in a significantly lower ratio of TI/TE in older foals. The allometric equation relating VT to bwt suggested that lung growth in the horse is dysanaptic, with increases in overall body size exceeding lung growth in the maturing foal during the first year post partum.

Changes in breathing pattern in the normal horse at rest up to age one year.

Changes in pattern of airflow, sequence of respiratory muscle activation and generated pressures were measured serially in a group of foals during the first year post partum, in order to describe the maturation of the equine breathing pattern. In neonatal foals, inspiration and expiration were both primarily active and airflow pattern was essentially monophasic. By age 1 year, foals displayed essentially the same breathing pattern previously described in adult horses, utilising a combination of active and passive inspiration and expiration to breathe around, rather than from, the relaxation volume of the respiratory system (Vrx). A strong temporal relationship during growth was found between the timing of changes observed in airflow pattern and in the neuromuscular strategy of breathing. The transition to the adult breathing pattern appeared to involve a time delay in activation of both inspiratory and expiratory muscle groups, establishing a passive and active component to both inspiration and expiration. Throughout the study period, concurrent with the increase in delay of abdominal muscle activation, the expiratory flow pattern became progressively more biphasic in appearance. The time of appearance of a consistent biphasic inspiratory flow pattern was considerably later, at approximately age 1 year and coincided with the appearance of a delay in inspiratory muscle activation. From our results, we conclude that the transition from the neonatal to the adult breathing strategy in the horse appears not to be induced by the time course of chest wall stiffening during maturation. While changes in relative body proportions and size of abdominal contents during growth may influence the transition in breathing, our results also indicate that respiratory control mechanisms play an essential role in the expression of the polyphasic breathing pattern.

Coagulation protein function. II. Influence of thiols upon acetaldehyde effects.

It has been reported that prolonged prothrombin time may be a result of the interaction of acetaldehyde (AcH) with clotting proteins to form alkylated inactive products. The current investigation focuses on the influence of L-cysteine (CysH), DL-homocysteine (HC), D-penicillamine, N-acetyl-L-cysteine (NAC), L-serine and L-alanine at 0.01 M concentrations, lactalbumin hydrolysate (2 mg/ml), and 1.0 mM dithiothreitol (DTT) on clotting time as well as their interaction with AcH. The sulfhydryl amino acids, as well as DTT prolonged clotting upon preincubation with plasma. Cysteine and NAC, upon addition to plasma prior to the addition of AcH, exhibited a prolongation of clotting time compared to that of AcH alone. On sequential addition of serine, alanine, or lactalbumin hydrolysate to plasma followed by the addition of acetaldehyde, a prolongation of clotting time comparable to that of AcH alone was exhibited. When HC and penicillamine were added to plasma prior to the addition of AcH, a prolonged clotting time was observed, which was significantly less than that of AcH alone. Premixing of serine, alanine, and lactalbumin hydrolysate with AcH for 20 min prior to addition to the plasma reduced the effectiveness of AcH in prolonging clotting time as compared to successive additions of the amino acid and AcH. Since CysH and penicillamine have been reported to form cyclic adducts with AcH, it is suggested that a similar possibility exists for penicillamine and for HC. The reversible cyclic adduct formation reported for CysH may explain why cysteine did not lower the prolonged clotting time induced by AcH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde alters coagulation protein function.

Acetaldehyde is the first metabolite of ethanol and has the potential to react with proteins and alter their function. This study evaluated the function of clotting proteins that had been preincubated with acetaldehyde as compared to those incubated with buffer or ethanol as controls. Thrombin, fibrinogen, thromboplastin, or whole plasma were preincubated with 1.8-447 mM acetaldehyde, 1.7-429 mM ethanol, or buffer for varying time periods prior to use in a clotting assay. Clot formation was measured with an automatic fibrometer. Acetaldehyde prolonged the clotting time but ethanol did not. These experiments indicate that circulating acetaldehyde would have the potential to react with proteins of the clotting system and alter their function. Therefore, it is possible that not all of the abnormalities in coagulation in alcohol abusers result from inadequate hepatic synthesis. Perhaps some of the deranged coagulation may be the result of the interaction of acetaldehyde with coagulation proteins.

Respiratory mechanics of the horse during the first year of life.

This study investigated the developmental changes in the mechanical properties of the respiratory system in growing horses. Pulmonary mechanics and lung volumes were serially measured in anesthetized foals during the first year of life. Quasi-static pressure-volume curves were generated, and functional residual capacity (FRC) was measured using a closed nitrogen equilibration technique. At birth, chest wall compliance normalized to body weight was substantially less than that reported in other less precocious newborn species, while lung compliance normalized to body weight was similar to values reported for other species. Characteristics of the transition from the neonatal to adult respiratory system in the foal included a decrease in the ratios of chest wall to lung compliance (Cw/CL) and the unstressed volume of the chest wall to TLC, and a constant FRC/TLC throughout most of the study period. The somatic growth of the foal and its respiratory system were uneven processes, with increases in lung volume lagging increases in overall body size.

High-frequency jet ventilation in a neonatal foal.

High-frequency jet ventilation was performed on a premature foal for respiratory difficulty attributable to in utero-acquired pneumonia. The procedure involves delivery of compressed gas through a small-bore cannula at frequencies up to 400 cycles/min. Ventilation settings of drive pressure, frequency, and FIO2 were varied to optimize PaO2 and PaCO2 values. The foal was ventilated with this equipment for 14 hours. Evidence of a favorable response to this method of ventilation was observed in the form of improvement in arterial blood gas values as well as the foal's attitude and degree of respiratory effort. High-frequency jet ventilation appears to be a useful method of ventilation for respiratory disease in neonatal foals; however, there remains no clear-cut advantage over conventional positive-pressure ventilation.

Breathing strategy of the adult horse (Equus caballus) at rest.

To investigate the mechanism underlying the polyphasic airflow pattern of the equine species, we recorded airflow, tidal volum, rib cage and abdominal motion, and the sequence of activation of the diaphragm, intercostal, and abdominal muscles during quiet breathing in nine adult horses standing at rest. In addition, esophageal, abdominal, and transdiaphragmatic pressures were simultaneously recorded using balloon-tipped catheters. Analysis of tidal flow-volume loops showed that, unlike humans, the horse at rest breathes around, rather than from, the relaxed volume of the respiratory system (Vrx). Analysis of the pattern of electromyographic activities and changes in generated pressures during the breathing cycle indicate that the first part of expiration is passive, as in humans, with deflation toward Vrx, but subsequent abdominal activity is responsible for a second phase of expiration: active deflation to below Vrx. From this end-expiratory volume, passive inflation occurs toward Vrx, followed by a second phase of inspiration: active inflation to above Vrx, brought about by inspiratory muscle contraction. Under these conditions the abdominal muscles appear to share the principal pumping duties with the diaphragm. Adoption of this breathing strategy by the horse may relate to its peculiar thoracoabdominal anatomic arrangement and to its very low passive chest wall compliance. We conclude that there is a passive and active phase to both inspiration and expiration due to the coordinated action of the respiratory pump muscles responsible for the resting adult horse's biphasic inspiratory and expiratory airflow pattern. This unique breathing pattern perhaps represents a strategy of minimizing the high elastic work of breathing in this species, at least at resting breathing frequencies.

Exploratory celiotomy for suspected urinary tract disruption in neonatal foals: a review of 18 cases.

The medical records of 18 neonatal foals, in which exploratory celiotomies were performed for suspected urinary tract lesions, were reviewed. Despite clinical signs and laboratory values indicative of disruption of the urinary tract, three foals did not have a site of urinary tract leakage at surgery. Eight foals had ruptured bladders and seven foals had urachal lesions. Ultrasonography was used as a pre-operative diagnostic procedure in eight foals to evaluate the presence of free peritoneal fluid and urinary tract integrity. Nine foals were alive six months after discharge. Seven of the nine non-surviving foals died or were destroyed because of fungal or bacterial infections.

Development of a scoring system for the early diagnosis of equine neonatal sepsis.

A sepsis scoring system was developed and tested prospectively in a blind study of 190 neonatal foals admitted to the University of Florida Veterinary Medical Teaching Hospital's neonatal intensive care unit. The system used 14 readily available historical, clinical or laboratory variables and weighted each item to arrive at a sepsis score. The score was found to have a sensitivity of 93 per cent, a specificity of 86 per cent, positive accuracy rate of 89 per cent and negative accuracy rate of 92 per cent. The sepsis score was far more sensitive and specific for infection, even in very early cases, and had fewer false positive and false negative values than did any parameter taken individually.

Exploratory celiotomy for gastrointestinal disease in neonatal foals: a review of 20 cases.

The medical records of 20 neonatal foals in which exploratory celiotomies were performed for gastrointestinal disease were reviewed. In all 20 foals, persistent pain and/or progressive abdominal distension were the primary clinical findings influencing the decision to operate. However, ancilliary laboratory data were important to the proper medical management of these foals during anaesthesia and following surgery. Surgical diagnoses of the 20 foals included ileus (nine foals; 45 per cent), small colon obstruction (five foals; 25 per cent), large colon displacement (three foals; 15 per cent), small intestinal displacement (two foals; 10 per cent), and perforated gastric ulcer (one foal; 5 per cent). Seventeen foals were recovered from anaesthesia, 13 of these were discharged from the hospital, seven were alive six months or more following discharge. Sepsis was the cause of death in six of the 10 foals that died following recovery.

Respiratory mechanics and breathing pattern in the neonatal foal.

Breathing pattern, respiratory muscle activation pattern, lung volumes and volume-pressure characteristics of the respiratory system of normal, term, neonatal foals on Days 2 and 7 of age were determined to test the hypothesis that the foal actively maintains end-expiratory lung volume (EEV) greater than the relaxation volume of the respiratory system (Vrx) because of a highly compliant chest wall. Breathing pattern was measured in the awake, unsedated foal during quiet breathing in lateral and standing positions. The typical neonatal foal breathing pattern was characterized by a monophasic inspiratory and expiratory flow pattern. Both inspiration and expiration were active, with onset of Edi activity preceding onset of inspiratory flow, and phasic abdominal muscle activity detectable throughout most of expiration. No evidence was found to support the hypothesis that the normal, term neonatal foal actively maintains EEV greater than Vrx. In the neonatal foal, normalized lung volume and lung compliance values were similar to those reported for neonates of other species, while normalized chest wall compliance was considerably lower. We conclude that the chest wall of the term neonatal foal is sufficiently rigid to prevent a low Vrx. This characteristic probably prevents the foal from having to use a breathing strategy which maintains an EEV greater than Vrx.