Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.).

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.

ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation.

The modulation of mitogen-activated protein kinase (MAPK) activity regulates many intracellular signaling processes. In animal and yeast cells, MAP kinases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase). Several plant homologs of MEK and MAPK have been identified, but the biochemical events underlying the activation of plant MAPKs remain unknown. We describe the in vitro activation of an Arabidopsis homolog of MAP kinase, ATMPK4. ATMPK4 was phosphorylated in vitro by an Arabidopsis MEK homolog, AtMEK1. This phosphorylation occurred principally on threonine (Thr) residues and resulted in elevated ATMPK4 kinase activity. A second Arabidopsis MEK isoform, ATMAP2Kalpha, failed to phosphorylate ATMPK4 in vitro. Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity. Immunoprecipitates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1. These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity of MAPK in plants. Surprisingly, in contrast to the animal enzymes, AtMEK1 may not be a dual-specificity kinase but, rather, the required Tyr phosphorylation on ATMPK4 may result from autophosphorylation.

Paget's disease of the vulva: prevalence of associated vulvar adenocarcinoma, invasive Paget's disease, and recurrence after surgical excision.

OBJECTIVE: Our aim was to determine the prevalence of associated vulvar adenocarcinoma, invasive Paget's disease, and recurrence of Paget's disease of the vulva. STUDY DESIGN: A retrospective review of tumor and pathology registries at 8 institutions is presented. Patients with recurrent disease were excluded. Histologic slide review was performed. RESULTS: The median age of the 100 patients was 70 years. The median duration of pruritus before surgery was 2 years. Thirty-four percent of patients experienced a recurrence at a median of 3 years. There was a 12% prevalence of invasive vulvar Paget's disease and a 4% prevalence of associated vulvar adenocarcinoma. One patient died of Paget's disease with associated vulvar adenocarcinoma. CONCLUSIONS: Paget's disease of the vulva is rarely associated with an underlying vulvar adenocarcinoma or invasive Paget's disease, but there is a high recurrence rate.

Epidermal cyst of the breast: detection in a screening programme.

Two cases of epidermal cyst of the breast, a rare benign condition, were detected during a 3-year period in a mammographic screening programme, from 57,954 screening examinations. It is not uncommon for epidermal cysts to be initially misdiagnosed. The mammographic, ultrasound and histological features are presented. It is recommended that these lesions be resected because they possibly have malignant potential.

Thyroid cancer complicating pregnancy.

Thyroid nodules and thyroid cancer are more common in women than in men. It is not uncommon to find a thyroid nodule during pregnancy that requires investigation. Fortunately, most cancers found during pregnancy are differentiated thyroid cancers with an excellent prognosis. Fine-needle aspiration is safe and accurate during pregnancy. Cancer discovered early in pregnancy can safely be operated on in the second trimester. Cancers or nodules discovered later in pregnancy can have work-up and treatment delayed until after delivery. No data support pregnancy termination or the proscription of future pregnancy in these patients.

Cloning and characterisation of MEK1, an Arabidopsis gene encoding a homologue of MAP kinase kinase.

We report the cloning of a cDNA for MEK1, an Arabidopsis thaliana gene encoding a homologue of MAP kinase kinase (MEK). The predicted protein sequence shows 41% identity over 270 amino acids to vertebrate MEK proteins, and contains conserved features characteristic of MEK. Analysis of transcript levels show that expression of the gene is regulated by developmental processes (etiolation/de-etiolation) and by wounding. However in contrast to the rapid wound induction of MAP kinase transcripts in other plant species, MEK1 transcripts first accumulated 6-12 h after wounding.

Osmotic lysis of tumor spill in ovarian cancer: a murine model.

OBJECTIVE: Our purpose was to determine, in the murine model, whether human ovarian cancer cells injected intraperitoneally are subject to osmotic lysis by peritoneal lavage with sterile water, thereby decreasing the establishment of peritoneal implants. STUDY DESIGN: Preliminary experiments on six nude mice determined that the injection of 20 million cells of the SKOV-3 cell line reliably leads to the establishment of intraperitoneal tumor xenografts in the mice within 60 days. Four other nude mice functioned as sham controls undergoing peritoneal lavage with 3 to 4 ml of saline solution or sterile water to determine any adverse effects from the lavage alone. Subsequently, 36 nude (nu/nu) mice were injected intraperitoneally with 1 ml of the SKOV-3 cell line at a concentration of 20 million cells per milliliter. Alternate mice then underwent intraperitoneal lavage with either 3 to 4 ml of normal saline solution (control group) or sterile water (study group). The mice were followed up until tumor growth caused a moribund status or until 60 days after injection and then were killed. At necropsy the number and size of tumor nodules were recorded, and each mouse was assigned a composite tumor score. Statistical comparison used the X2 or Fisher's exact test for discrete variables. Time to failure analysis used the Kaplan-Meier method. RESULTS: Tumor growth occurred in 35 of 36 (97%) of the mice during the study period. In the first 30 days 89% of the saline solution group grew clinically visible tumor compared with 55% of the water group (p = 0.03). Ascites developed more frequently in the water group than in the saline solution group. The median tumor scores at death were significantly higher for the water group versus the saline solution group. Survival time, as determined by the time from injection until moribund status, was worse for the water group (p = 0.002). CONCLUSIONS: Intraperitoneal lavage with sterile water did not offer protection against the establishment of xenografts after the intraperitoneal injection of human ovarian cancer cells in the nude mouse model.

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

Intraoperative autologous blood collection and autotransfusion in the surgical management of early cancers of the uterine cervix.

OBJECTIVES: To evaluate intraoperative autologous blood collection with autotransfusion (Cell Saver) with respect to patient acceptance, risk of tumor cell co-transfusion, and risk of recurrence in patients undergoing radical hysterectomy for cervical cancer. METHODS: All patients explored for radical hysterectomy between August 1991 and July 1994 were offered the use of intraoperative autotransfusion. Clinical-pathologic and transfusion-related characteristics were compared for a group of historic controls surgically treated for similar disease. The risk of tumor cell co-transfusion was assessed intraoperatively with peritoneal cytology before blood collection, and postoperatively with Cell Saver blood cytology. RESULTS: Ninety-eight patients were offered enrollment; four declined Cell Saver use, and 71 were acceptable for analysis. Thirty-one women (mean estimated blood loss 1338 mL) were reinfused with their own blood collected in the Cell Saver, whereas 40 patients (mean estimated blood loss 631 mL) were not autotransfused. There was no significant difference in preoperative hemoglobin concentration between groups. Cell Saver use significantly reduced the need for homologous transfusions, intraoperatively (P < .001) and postoperatively (P = .02). Historic controls (mean operative blood loss 1743 mL) were nearly four times more likely to have been transfused and three times more likely to have been transfused postoperatively than was the auto-transfused Cell Saver group. The mean hemoglobin concentration at discharge was lower in the autotransfused group, 9.3 g/dL, than in the historic controls, 10.8 g/dL. Nontransfused Cell Saver blood and all peritoneal cytologies were negative for tumor cells. Three pelvic recurrences, but no disseminated disease, have been noted over a mean follow-up of 24 months: one in the autotransfused group and two in the group in which the collected blood was discarded. CONCLUSION: Cell Saver use is well accepted by patients, decreases the need for homologous transfusions, and does not appear to co-transfuse tumor cells.

Blood transfusion and the risk of recurrence in stage IB cervical cancer.

The objective of this study is to determine if perioperative blood transfusions increase the risk of recurrence in stage IB cervical cancer. Medical records from all patients with FIGO stage IB cervical cancer undergoing radical hysterectomy and pelvic lymphadenectomy (RH + PLND) at the University of Iowa and the University of Nebraska from 1978 to 1990 were retrospectively reviewed. Data collected included patient age, body mass index (BMI), tumor size, cell type, depth of cervical invasion (DOI), presence of capillary-lymphatic space involvement (CLSI), lymph node metastasis, operating time, estimated blood loss, transfusion, and follow-up data. Three hundred two patients underwent RH + PLND. Transfusions were given to 244 (81%), with a mean of 2.6 units (range 1-18 units). Median follow-up was 49.5 months (range 9-190 months). Twenty patients (6.6%) had pelvic nodal metastasis. There were no periaortic nodal metastases in the 101 patients who had periaortic nodes dissected. There were no significant differences between the transfused and nontransfused groups, with respect to age, BMI, DOI, or pelvic node metastasis. Transfused patients differed significantly from the nontransfused in that they had larger tumors (P = 0.047), more frequent CLSI (P = 0.013), longer procedures (P = 0.02), and greater estimated blood loss (P < 0.0001). Recurrences developed in 29 patients (19 pelvic, 7 lung, 3 bone). There is no difference in disease-free survival (DFS) or calculated projected survival between the transfused and nontransfused groups. Pelvic node metastasis and tumor size were independent poor prognosticators. After controlling for these factors, the number of blood transfusions was not predictive of recurrence or survival. Perioperative transfusions do not increase the risk of recurrence in patients with cervical cancer.

Steroid hormone receptor content and lymph node status in endometrial cancer.

The purpose of this study was to correlate the steroid hormone receptor status in endometrial adenocarcinoma with tumor metastasis to the pelvic and para-aortic lymph nodes, and with other known prognostic variables which influence survival. Tumor samples from 85 patients with endometrioid adenocarcinoma, or adenocarcinoma with squamous differentiation of the endometrium who underwent complete surgical staging, were assayed for cytoplasmic steroid hormone receptors using a dextran-coated charcoal technique. Steroid hormone receptor content was correlated to lymph node status, along with other prognostic variables, such as patient's age, depth of myometrial invasion, tumor grade, and pelvic cytology. By univariate analysis, the likelihood of nodal involvement was associated with younger age and poorly differentiated tumors. Multivariate analysis revealed that age, tumor grade, and myometrial involvement added significant independent prognostic information. Estrogen or progesterone receptor content did not add independent prognostic information concerning lymph node status once other factors were controlled. Knowledge of estrogen and progesterone receptor binding status in adenocarcinoma of the uterus does not replace the prognostic information imparted by careful sampling of lymph nodes.

Current advances in abscisic acid action and signalling.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.

The Arabidopsis ABI1 locus is essential for a wide spectrum of abscisic acid (ABA) responses throughout plant development. Here, ABI1 was shown to regulate stomatal aperture in leaves and mitotic activity in root meristems. The ABI1 gene was cloned and predicted to encode a signaling protein. Although its carboxyl-terminal domain is related to serine-threonine phosphatase 2C, the ABI1 protein has a unique amino-terminal extension containing an EF hand calcium-binding site. These results suggest that the ABI1 protein is a Ca(2+)-modulated phosphatase and functions to integrate ABA and Ca2+ signals with phosphorylation-dependent response pathways.

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement.

Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.

Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana.

Gene tagging in Arabidopsis thaliana using the autonomous Ac (Activator) transposable element has so far been hampered by low frequencies of germinal transposition events. Here we describe a procedure by which the frequency of independent germinal reinsertions has been much improved by a process of long-term selection on kanamycin for the continued growth of tissues in which somatic excisions have occurred. Growth on artificial media increased the somatic excision frequency, and the long-term selection procedure channelled somatic transposition events into the germline. This resulted in an overall germinal excision frequency in the progeny of longterm selected plants of 15%, as confirmed by Southern blotting, with 63% of the plants bearing excision events having detectable reinsertions of the Ac element. This compares with a germinal excision frequency of approximately 1% when no long-term selection is employed. However, offspring from individual plants tended to have identical germinal Ac reinsertion patterns, thus the critical parameter for evaluating the system for tagging purposes is the frequency of individual plants yielding offspring with reinsertions, which was 64%. This high frequency, when coupled to the enhanced germinal transposition rate overall, easily allows the generation of a large population of plants with independent reinsertions.

Comparison of clinical versus surgical staging systems in vulvar cancer.

OBJECTIVE: To compare prognostic information from the new surgical staging system of the International Federation of Gynecology and Obstetrics (FIGO) with the old clinical staging system for vulvar cancer. METHODS: One hundred six women with previously untreated squamous cell carcinoma of the vulva who underwent radical vulvectomies and inguinal lymph node dissections at the University of Oklahoma from 1971-1990 were considered eligible for this study. A retrospective chart review was conducted to assign surgical stage. The clinical and pathologic factors analyzed for survival included the clinical and surgical stage of disease, nodal status, tumor size, and lesion location. RESULTS: Overall 5-year survival was 64%. Forty-three patients had inguinal and femoral node metastasis with a 5-year survival of 38%, versus 87% for patients without nodal metastasis (P < .00001). An increased number of positive groin lymph nodes was associated with a poorer prognosis. Thirty-one patients had tumors of 2 cm or less in maximum diameter with no recurrences, versus 52% 5-year survival in the remaining patients (P < .001). Perineal involvement was identified in 24 patients, but did not significantly influence survival. CONCLUSION: Overall, the new classification system revised by FIGO for vulvar cancer staging places patients into more accurate risk categories.

The significance of peritoneal cytology in stage IB cervical cancer.

OBJECTIVE: The purpose of this study was to determine the incidence of positive peritoneal cytology and to evaluate its usefulness in the management of patients with early-stage cervical cancer. METHODS: Peritoneal cytology was studied in 273 women undergoing primary surgical exploration for International Federation of Gynecology and Obstetrics stage IB cancer of the cervix. Charts were reviewed retrospectively for clinicopathologic data concerning tumor size, cell type, lymph node status, and outcome. RESULTS: Cytology was positive in four women, three of whom had enlarged pelvic or para-aortic lymph nodes or intraperitoneal disease. There was no association between tumor histology or tumor size and peritoneal cytology. CONCLUSION: The incidence of positive peritoneal cytology in early-stage cervical cancer is low, and the prognostic significance of positive cytology is overshadowed by other risk factors more obvious at surgery. The routine collection of cytologic specimens at laparotomy should be abandoned in this setting.

Gene targeting in Arabidopsis thaliana.

Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10(8) protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10(-4).

Osmotic stress and abscisic acid induce expression of the wheat Em genes.

The early-methionine-labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination. Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em-gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes.

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.