Biomedical subjects

T Kawata

Publications and source records attributed to T Kawata.

At least 73 records · Page 4Linked to original sources

PubMed2000

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Animals↗

PubMed2000

High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts.

PURPOSE: To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS: The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS: Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.

Cell Line↗

PubMed2000

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2000

Effect of cobalamin on the allergic response in mice.

Cyanocobalamin, deoxyadenosylcobalamin and methylcobalamin were separately administered to mice sensitized with the ovalbumin antigen to investigate the allergic response. The serum IgE and pulmonary histamine concentrations were significantly lower in all cobalamin (Cbl)-administered groups. The production of interleukin (IL)-2 and IL-4 in splenocytes was also lower in all Cbl-administered groups. These results show that Cbls were effective in mitigating allergic reactions and IL-2 production. CD3+ CD28+ (CD28 is an accessory molecule related to IL-2 production) and CD4+ CD28+ in splenocytes were higher in all the Cbl-administered groups. However, CD3+ CD28-, CD4+ CD28- and CD5+ CD25- (CD25: IL-2 R alpha/p55) were lower in the Cbl-administered groups. In addition, Cbl specifically inhibited the cellular phosphorylation of tyrosine induced by ovalbumin sensitization. These results indicate that the signal in a cell by CD 28 was restrained by Cbl. We infer that Cbl administration significantly reduced the IL-2 concentration, and secondarily the IL-4, IgE and histamine concentrations.

Animals↗

PubMed1999

Induced expression, localization, and chromosome mapping of a gene for the TBP-interacting protein 120A.

TBP-interacting protein 120A (TIP120A) is a novel eukaryotic transcriptional regulator and has been suggested to be involved in the general regulation of transcription because of its ability to potentiate transcription of all classes of genes and to interact with common transcriptional machineries. In the present study, we investigated the expression of the tip120a gene. TIP120A transcripts were expressed abundantly in the heart and liver, moderately in the brain and skeletal muscle, and only slightly in the spleen and lung. This ubiquitous expression pattern was similar to that of TBP. Gene expression of TIP120A in the rat liver was not stimulated by hepatocarcinogenesis or liver regeneration. TIP120A was thus suggested not to be a growth-related protein. On the other hand, in P19 mouse embryonal carcinoma cells, TIP120A expression was elevated upon retinoic acid treatment, which induces differentiation. Notably, the foci-like nuclear localization pattern of TIP120A was transformed into a speckle-like pattern. The level of TIP120A was also elevated in such stem-like cells as F9 and HL60 after each differentiation procedure, retinoic acid and DMSO, respectively. In HEp-2 cells, TIP120A was observed as a limited number of nuclear foci, and the localization coincided with that of the PML oncogenic domain. FISH detection revealed that the human tip120a gene was located at 12q14, the position to which a myopathic type scapuloperoneal syndrome locus also mapped. Our study suggests that, contrary to an early assumption, TIP120A is involved in tissue-specific and/or differentiation-related gene expression.

Animals↗

PubMed1999

Analysis of the chicken TBP-like protein(tlp) gene: evidence for a striking conservation of vertebrate TLPs and for a close relationship between vertebrate tbp and tlp genes.

TLP (TBP-like protein), which is a new protein dis-covered by us, has a structure similar to that of the C-terminal conserved domain (CCD) of TBP, although its function has not yet been elucidated. We isolated cDNA and genomic DNA that encode chicken TLP (cTLP) and determined their structures. The predicted amino acid sequence of cTLP was 98 and 91% identical to that of its mammalian and Xenopus counterparts, respectively, and its translation product was ubiquitously observed in chicken tissues. FISH detection showed that chicken tlp and tbp genes were mapped at 3q2.6-2.8 and 3q2.4-2.6 of the same chromosome, respectively. Genome analysis revealed that the chicken tlp gene was spliced with five introns. Interestingly, the vertebrate tbp genes were also found to be split by five introns when we focused on the CCDs, and their splicing points were similar to those of tlp. On the contrary, another TBP-resembling gene of Drosophila, trf1, is split by only one intron, as is the Drosophila 's tbp gene. These results support our earlier assumption that vertebrate TLPs did not directly descend from Drosophila TRF1. On the basis of these results together with phylogenetical exam-ination, we speculate that tlp diverged from an ancestral tbp gene through a process of gene duplication and point mutations.

Amino Acid Sequence↗

PubMed1999

Late retinal complications of radiation therapy for nasal and paranasal malignancies: relationship between irradiated-dose area and severity.

PURPOSE: Radiation-induced cataract, once a notorious ocular complication of radiation therapy, is no longer considered a severe complication, because visual acuity can be restored by surgical treatment without significant complications. Late retinal complications of retinopathy and glaucoma, for which there is no effective method of treatment, have become serious complications of radiotherapy of the head and neck. We retrospectively investigated the risk of late retinal complications of radiotherapy for nasal and paranasal malignancies according to the radiation dose and area of the retina irradiated. METHODS AND MATERIALS: Between October 1982 and May 1996, 43 eyes of 25 patients were exposed to fractionated external-beam irradiation for treatment of advanced nasal and paranasal cancer. None of the patients had tumor invasion into the eyes. The patients were followed ophthalmologically for a minimum of 2 years (range 2.0-11, mean 4.5, median 3.3). The radiation dose and area of the retina irradiated were estimated from the dose distribution figures calculated using the portal films and CT scan. RESULTS: Major late adverse effects of radiotherapy were observed in the retina in 9 of 43 eyes (in 8/25 patients). Radiation retinopathy was observed in 7 eyes, and the cumulative incidence was 25%. The median interval before the onset of symptoms attributable to retinopathy was 32 months (range 16-60). Neovascular glaucoma developed in 3 of the 43 eyes, with a cumulative incidence of 7%. The median period to the onset of symptoms attributable to glaucoma was 22 months (range 16-26). Obstruction of the central retinal artery was observed in 1 eye. The irradiation doses to the retinas that developed late complications ranged between 54-75 Gy (mean 61, median 61). No patients who received less than 50 Gy developed retinal complications. The retina in 21 eyes was exposed to a dose of 50 Gy or more. In 13 of the 21 eyes, 60% or more of the retina was irradiated, and 8 of the eyes (62%) in this group (> or = 50 Gy, > or = 60%) developed severe retinal complications, whereas such complications only developed in 1 of the 8 eyes (13%) in the other group (> or = 50 Gy, > or = 60%). The results suggest that the radiation dose and area irradiated are the most important factors in the development of severe complications. CONCLUSION: Radiation-induced retinopathy and glaucoma are more serious late complications than cataracts, which are easily treated with surgery. We investigated the risk of late retinal complications of radiotherapy, and our findings suggested that the radiation dose and area irradiated are the most important factors in the development of severe complications. We recommend that the radiation dose and area of the retina irradiated be minimized in patients at risk of eye complications, and the patients should be closely followed by periodic ophthalmologic testing after treatment.

Adult↗

PubMed1999

Isolation of cDNA, chromosome mapping, and expression of the human TBP-like protein.

TBP is an essential factor for eukaryotic transcription. In this study, we identified a human cDNA encoding 21-kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of human TBP with 39% identity and 76% similarity. FISH determined that human tlp gene was located at chromosome 6 region q22.1-22.3. Northern blot analysis demonstrated that TLP mRNAs were expressed in various human tissues ubiquitously. We found that the TLP proteins exist in multiple mammalian cells and chicken cells. Although the Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor, expression of TLP was nearly constant throughout the neural differentiation of P19 cells. Unlike TRF, TLP did not bind to the TATA-box nor direct transcription initiation in vitro. Similarity between TRF and TLP was considerably lower (35 in alignment score) than that between Drosophila TBP and human TBP (88 in alignment score). Multiple amino acids critical for the TBP function were deleted or substituted in TLP. We suggest that TLP is not a bona fide vertebrate counterpart nor a direct descendant of TRF.

Amino Acid Sequence↗

PubMed1999

Benefits accruing to grafting of the right internal thoracic artery to the left anterior descending artery in coronary artery bypass grafting.

OBJECTIVES: We have investigated the effects and outcome from grafting the right internal thoracic artery to the left anterior descending artery in coronary artery bypass grafting. METHODS: We analyzed the findings in 185 patients who underwent coronary artery bypass grafting involving right internal thoracic artery-left anterior descending artery bypass. The survival rate, cardiac events related to the right internal thoracic artery-left anterior descending artery graft, and graft patency were investigated. In cardiac catheterization, the production of nitric oxide from the endothelium of the internal thoracic artery was measured as the plasma nitrite and nitrate levels by the Griess reaction. The reactions to acetylcholine infusion (5 micrograms) in the right internal thoracic artery (n = 4) were compared to those in the left internal thoracic artery (n = 9) grafts. RESULTS: The hospital mortality rate was 0.5%. The actuarial survival rate at 11 years after operation was 98.81%. The rate of freedom from cardiac events at 11 years after operation was 92.08%. The rate of patency of the right internal thoracic artery-left anterior descending artery was 97.0% in 133 patients. Nitric oxide production was calculated as (total nitrite plus nitrate production in response to stimulation by acetylcholine-total nitrite plus nitrate level before acetylcholine load)/(total nitrite plus nitrate level before acetylcholine load). There was no significant difference in changes in plasma concentration in response to acetylcholine stimulation between the right internal thoracic artery-left anterior descending artery and left internal thoracic artery-left anterior descending artery groups (20 +/- 29% versus 5 +/- 10%). CONCLUSIONS: The right internal thoracic artery-left anterior descending artery grafts exhibited good patency and release of nitric oxide in response to acetylcholine loading, similar to left internal thoracic artery-left anterior descending artery grafts.

Coronary Artery Bypass↗

PubMed1999

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai).

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0 to approximately 300 microl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (> or = 10 microl/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 microl/kg f.w./h-10 microl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear.

Amino Acid Oxidoreductases↗

PubMed1999

Minimally invasive direct coronary artery bypass combined with abdominal aortic aneurysm repair.

BACKGROUND: For simultaneously combined coronary artery bypass surgery with infrarenal abdominal aortic aneurysm (AAA) repair, a relatively high operative mortality and morbidity have been reported. METHODS: From February 1998 to December 1998, simultaneous minimally invasive direct coronary artery bypass combined with the AAA repair was performed for 4 patients (3 males, 1 female; mean age, 74+/-7 years). Three were high-risk patients: 2 were over 75 years of age, 2 had respiratory insufficiency, and 1 had severe renal impairment. RESULTS: There were no mortalities. The endotracheal tube was removed within approximately 12 hours, and the postoperative courses were uneventful. During 4+/-4 months of follow-up, there was neither angina recurrence nor other morbidity. CONCLUSIONS: Minimally invasive direct coronary artery bypass combined with AAA repair was safe even for high-risk patients.

Aged↗

PubMed1999

Recruitment of osteoclasts in the mandibular condyle of growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor.

The purpose was to elucidate histological changes in the mandibular condyle and ramus in growing osteopetrotic (op/op) mice after a single injection of macrophage colony-stimulating factor (M-CSF). M-CSF (5 microg) was injected into 6-, 11-, 26-, 56- and 86-day-old op/op mice, and the mice were killed 4 days after the injection. In normal mice, the condyle was substantially wider than the ramus beneath it, and enlargement and ossification of the condyle occurred after weaning. These changes were not found in the uninjected and injected op/op mice, the condyles of which were occupied by hypertrophic cartilage cells, and the hypertrophic cell layer was thicker and more irregular in the arrangement of epiphyseal cell columns. In spite of the lack of bone resorption in uninjected and injected op/ op mice, ossification of the mandibular ramus occurred, but later than that of normal mouse. The number of tartrate-resistant acid phosphatase-positive cells in the injected op/op and normal mice approached a maximum at 30 days and then gradually decreased up to 90 days of age, although the numbers were substantially different for all ages. The uninjected op/op mice had no visible osteoclasts until 15 days and their number then increased significantly from 60 to 90 days of age. These results were considered due to the difference in biological responses of bony structures to M-CSF injection in the op/op mice. The influences of mechanical stimuli from masticatory functions, which are deficient in op/op mice, might also be responsible for the differences in bony architecture between the op/op and normal mice.

Animals↗

PubMed1999

E5531, a synthetic non-toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide.

1. The major pathological responses to Gram-negative bacterial sepsis are triggered by endotoxin or lipopolysaccharide. As endotoxin is shed from the bacterial outer membrane, it induces immunological responses that lead to release of a variety of cytokines and other cellular mediators. As part of a program aimed at developing a therapeutic agent for septic shock, we have developed E5531, a novel synthetic lipopolysaccharide antagonist. 2. As measured by release by tumour necrosis factor-alpha, human monocytes or whole blood can be activated by lipopolysaccharide, lipid A, and lipoteichoic acid (from Gram-positive bacteria). E5531 potently antagonizes activation by all these agents while itself being devoid of agonistic activity. 3. The inhibitory activity of E5531 was dependent on time of addition. When 10 nM E5531 was added simultaneously with lipopolysaccharide or 1 - 3 h before addition of lipopolysaccharide, production of tumour necrosis factor-alpha was inhibited by more than 98%. The addition of E5531 1 h after lipopolysaccharide reduced the efficacy of E5531 by 47%. 4. Antagonistic activity of E5531 was specific for lipopolysaccharide as it was ineffective at inhibiting interferon-gamma mediated NO release of RAW 264.7 cells, phorbor 12-myristate 13-acetate stimulated superoxide anion production in human neutrophils, concanavalin A stimulated mitogenic activity in murine thymocytes and tumor necrosis factor-alpha induced E-selectin expression in human umbilical vein endothelial cells. 5. E5531 as well as MY4, an anti-CD14 antibody, inhibited radiolabelled lipopolysaccharide binding in human monocytes. 6. These results support our contention that E5531 is a potent antagonist of lipopolysaccharide-induced release of tumour necrosis factor-alpha and other cellular mediators and may be an effective therapeutic agent for human septic shock due to Gram-negative bacteria.

E-Selectin↗

PubMed1999

Influences of vertical occlusal discrepancies on condylar responses and craniofacial growth in growing rats.

The present study was conducted to investigate the influence of vertical occlusal discrepancies on condylar remodeling and the subsequent craniofacial growth in growing rats. Thirty 4-week-old male Wistar strain rats were used. A 1-mm-thick metal plate was bonded onto the occlusal surface of the maxillary molars to increase posterior dentoalveolar height. During the early phase of the experiment, the thickness of the proliferative and maturative/hypertrophic zones in the anterior and superior portions of the condyle was significantly smaller in the experimental group than in the controls. The number of TRAP-positive cells was significantly greater in the experimental group than in the controls. At the end of the experiment, decreased ramus height and a large gonial angle were found in the experimental group. Changes in the intra-articular environment associated with vertical occlusal discrepancies may influence condylar and craniofacial growth in growing individuals, although some adaptive response of the condyle may be induced if growth potential remains.

Acid Phosphatase↗

PubMed1999

Reconsideration of the TMJ condylar position during internal derangement: comparison between condylar position on tomogram and degree of disk displacement on MRI.

The objective of this study was to investigate condylar position during different degrees of disk displacement. The degree of disk displacement was evaluated on 1.5 Tesla high-field sagittal MR images of 76 joints (48 patients; mean age 19.4 years) and was classified into three grades (1 to 3). To establish condylar position, the anterior, superior and posterior joint spaces were measured on corrected tomograms. The possible relationship between condylar position, indicated by the width of joint space and successive degree of disk displacement, was compared by a one-way factorial ANOVA (p < 0.05). The anterior joint space was significantly larger in grades 1, 2, and 3 disk displacements than in grade 0 pointing to a dorsal condylar position. The posterior joint space was significantly smaller in grades 1 and 2 compared with grade 0. When the degree of the disk displacement becomes severe (i.e., grade 3 disk displacement), the posterior joint space tends to increase to a similar distance as in a normal healthy joint pointing to a more normal condylar position.

Adolescent↗

PubMed1999

Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation.

PURPOSE: To analyse the kinetics of chromatid break induction, rejoining, and misrejoining after y-irradiation in G2 phase human cells using premature chromosome condensation induced by calyculin A. MATERIALS AND METHODS: Human fibroblast AG1522 cells were irradiated with gamma-rays and chromosomes were then prematurely condensed by calyculin A. The number of chromatid breaks and chromatid exchanges in G2 chromosomes were scored, and fitted curves were calculated. RESULTS: Calyculin A induced premature chromosome condensation in cells immediately after irradiation. Kinetics of rejoining of chromatid breaks demonstrated two exponential components with rapid and slow time constants. Within 5 min after irradiation, the number of chromatid breaks fell rapidly to about one-half, then gradually decreased. Chromatid exchanges were formed very quickly, reaching a plateau within 20 min from exposure. CONCLUSIONS: Chemically induced premature chromosome condensation technique allows a simple, rapid and precise analysis of chromatid breakage and rejoining. The rapid kinetic component was particularly well characterized.

Cells, Cultured↗

PubMed1999

Measurements of the equivalent whole-body dose during radiation therapy by cytogenetic methods.

Estimates of equivalent whole-body dose following partial body exposure can be performed using different biophysical models. Calculations should be compared with biodosimetry data, but measurements are complicated by mitotic selection induced in target cells after localized irradiation. In this paper we measured chromosomal aberrations in peripheral blood lymphocytes during radiotherapy, and estimated the equivalent whole-body dose absorbed, by using the novel technique of interphase chromosome painting. Premature chromosome condensation was induced in stimulated lymphocytes by incubation in calyculin A, and slides were hybridized in situ with whole-chromosome DNA probes specific for human chromosomes 2 and 4. Reciprocal exchanges were used to estimate the equivalent whole-body dose, based on individual pre-treatment in vitro calibration curves. Equivalent whole-body dose increased as a function of the number of fractions, and reached a plateau at high fraction numbers. Chromosomal aberration yields were dependent on field size, tumour position and concurrent chemotherapy. Results suggest that interphase chromosome painting is a simple technique able to give a reliable estimate of the equivalent whole-body dose absorbed during therapeutic partial-body irradiation.

Aged↗

PubMed1999

Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

8-Bromo Cyclic Adenosine Monophosphate↗