Activator protein 1 transcription factors are fundamental to v-rasHa-induced changes in gene expression in neoplastic keratinocytes.

The induction of mouse skin papillomas by initiation-promotion protocols is associated with aberrant expression of epithelial markers in the tumor mass. Similarly, initiation of mouse keratinocytes with a retrovirus encoding the v-rasHa gene (v-rasHa keratinocytes) causes characteristic alterations of epidermal gene expression (A. A. Dlugosz et at, Cancer Res., 54: 6413-6420, 1994). Because activator protein 1 (AP-1) proteins are likely targets of Ras activation, we have examined the role of AP-1 factors in v-rasHa keratinocytes. Introduction of v-rasHa into keratinocytes up-regulates c-Fos, deltaFos B, and Fra-1 transcripts and protein levels in nuclear extracts. The expression of Jun proteins is not significantly altered in v-rasHa keratinocytes. Transduction of cells with v-rasHa results in increased AP-1-dependent transcriptional activity, which is also simulated by transfection of keratinocytes with either c-Fos or deltaFos B but not Fra-1, suggesting that the up-regulation of c-Fos and deltaFos B contributes to this effect. To explore the role of AP-1 proteins in regulating keratinocyte markers in v-rasHa keratinocytes, we blocked the binding of AP-1 proteins to DNA by infecting keratinocytes with an adenovirus encoding a dominant-negative Fos mutant (A-FOS). A-FOS replaces endogenous Fos proteins in the formation of heterodimers with Jun family members and thus prevents the AP-1 transcription factor from binding to DNA. In v-rasHa keratinocytes, the A-FOS virus reversed the suppression of keratins 1 and 10 transcripts and protein, which is characteristically seen in tumors and v-rasHa keratinocytes. A-FOS also increased protein levels but reduced transcripts for the late marker, loricrin, a component of the cornified envelope. These findings indicate that AP-1 proteins are involved in the changes in gene expression that define the v-rasHa phenotype in mouse keratinocytes.

CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes.

Previously, we have shown that nuclear extracts from cultured mouse keratinocytes induced to differentiate by increasing the levels of extra-cellular calcium contain Fra-1, Fra-2, Jun B, Jun D and c-Jun proteins that bind to the AP-1 DNA binding sequence. Despite this DNA binding activity, AP-1 reporter activity was suppressed in these cells. Here, we have detected the CREB family proteins CREB and CREMalpha as additional participants in the AP-1 DNA binding complex in differentiating keratinocytes. AP-1 and CRE DNA binding activity correlated with the induction of CREB, CREMalpha and ATF-1 and CREB phosphorylation at ser133 (ser133 phospho-CREB) in the transition from basal to differentiating keratinocytes, but the activity of a CRE reporter remained unchanged. In contrast, the CRE reporter was activated in the presence of the dominant-negative (DN) CREB mutants, KCREB and A-CREB, proteins that dimerize with CREB family members and block their ability to bind to DNA. The increase in CRE reporter activity in the presence of these mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor that dimerizes with the CREB leucine zipper. In experiments where the A-CREB mutant was co-transfected with an AP-1 reporter construct, transcriptional activity was also increased indicating that a CREB family member binds AP-1 sites and represses AP-1 transcriptional activity as well. Exogenous expression of the transcriptional repressor CREMalpha down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contribute to the suppression of AP-1 transcriptional activity observed in differentiating keratinocytes.

Curriculum integration in nutrition and mathematics.

Today's school-aged children face a multitude of health issues that affect their well-being and academic performance. Partnerships have developed between health and education agencies to help American children succeed at math and science and to prepare them to make healthful, lifelong decisions. Curriculum integration provides a framework for children to apply knowledge from several disciplines and to use this knowledge to solve real-life problems at work and at play. Goals for instruction focus on the needs not only of the individual but also of society. Nutrition science and mathematics form a natural partnership. Nutrition science incorporates numerous mathematical concepts and procedures such as sorting, classifying, statistics, probability, estimation, and rates and proportion. In preparation for participation in a global and technological society that will require citizens to be quantitative thinkers, educators must endeavor to assist all children in becoming adults who are mathematically literate and competent.

Surveys yield data on practice overhead, compensation, and productivity.

Results of three separate surveys conducted in 1996 on group practice costs, management and physician compensation, and productivity showed surprising results. Family practice has the highest overhead of any specialty. Medical practices seem willing to pay premium rates to hire experienced managed care administrators and/or those with large medical practice experience. And even though specialty physicians' productivity is increasing faster than that of primary care physicians, the primary care physicians' pay is increasing faster.

A new procedure for increasing efficiency of protoplast plating and clone selection.

Sea Prep(TM) agarose (FMC corp.) was used as a gelling agent for media in the culture of potato and tomato protoplasts. The concentration and conditions appropriate for gelling and liquifaction were determined. The new method provided increased calli transfer efficiency of up to 50-fold. Shoot formation from the protoplast-derived cells occurred normally and without reduction in frequency.

Selection and characterization of cadmium-resistant suspension cultures of the wild tomato Lycopersicon peruvianum.

Suspension cultures of Lycopersicon peruvianum were selected for resistance to cadmium by stepwise exposure to increasing concentrations of cadmium sulfate. Resistant cells grow in 1500 micromolar Cd(++). This resistance was retained for thirty generations without selection. Both resistant and parental sensitive cultures take up Cd(++) at similar rates and to the same final levels. Exposure of sensitive or resistant cultures to Cd(++), Cu(++), or Zn(++) leads to the intracellular accumulation of a low molecular weight, cysteine-rich, cadmium-binding protein. This metallothionein is induced over fifteen fold by 100 µM cadmium and builds up to about five fold higher levels in the resistant cultures.

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep.

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized Ascaris suum antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A2, prostaglandin (PG) D2 and PGI2 from the PG endoperoxide intermediate, PGH2, was assayed over a range of substrate concentrations from 3-200 microM. Synthesis of PGI2 by trachealis microsomes was approximately 5-fold greater than that of TXA2. PGI2 and TXA2 production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA2 production predominated over PGI2 by 9-fold, preparations from Ascaris-exposed animals synthesized 50% more TXA2 than controls at PGH2 concentrations of 25 microM and above, whereas synthesis of PGI2 and PGD2 were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA2 and PGI2 in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA2 synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.

Developing a clinical competency examination in radiology: part I--test structure.

BACKGROUND: There is a need for radiology educational outcome measures that evaluate clinical competency. This is the first of two articles introducing a clinical competency examination in radiology. This first article describes the evolution and structure of the examination. The second article presents the results of two administrations of the examination and evaluates possible outcome predictors. OBJECTIVE: To develop a measure of clinical competency in radiology. DESIGN: Descriptive. METHODS: A test was developed to simulate the radiologic interpretive skills needed in clinical chiropractic practice. Students were timed as they responded to questions regarding the localization, categorization, management, and identification of pathologic conditions presented on plain film images of the spine and chest. Twenty-five radiographic cases were displayed at individual viewbox stations, and student responses to the 4 questions were compared with the consensus responses of 2 radiology instructors. RESULTS: Two versions of the test, using different cases, were given to the same ninth trimester class of 210 chiropractic students in a 10-trimester program. Of the 210 students, 116 (55.2%; 86 men and 30 women) took version 1 of the examination, and 1 month later 181 (86.2%: 138 men and 43 women) took version 2 of the examination. Student comments after each examination were favorable. CONCLUSION: The test structure uniquely provides information related to the student's ability to localize, categorize, manage, and identify pathologic conditions on imaging studies. At present the examination is instructor time intensive; further refinement is needed before wide implementation can occur.

Developing a clinical competency examination in radiology: part II--test results.

BACKGROUND: This is the second of two articles introducing a clinical competency examination in radiology. The first article described the structure, administration, and postexamination student comments for two versions of the radiology competency examination. This article reports the results obtained from these two administrations of the examinations. OBJECTIVE: To measure and identify potential outcome predictors of student aptitude in clinical film interpretation. DESIGN: Experimental. METHODS: An examination was developed to simulate the radiologic interpretive skills needed in clinical chiropractic practice. Two versions of the examination were given to a class of 210 ninth trimester students in a 10-trimester chiropractic program. Linear regression and bivariate correlations were performed on possible predictors of student success and test scores on the version 2 examination. RESULTS: On version 1 of the examination, students were able to identify an average of 59.6% of the normal cases as normal and 51.6% of abnormal cases as abnormal. On version 2, 55.6% of the normal cases were recognized as normal and 58.2% of abnormal cases as abnormal. On both versions, students were less successful at correctly categorizing, managing, or naming pathologic conditions they found. Of the predictors evaluated, only the students' grades in the third radiology course (tumors, arthritides, and extremity trauma) and the scores on the diagnostic imaging section of National Boards part II were significant predictors. DISCUSSION: Our results should cause some concern for educators who use content-based radiology curricula. Students demonstrated poor abilities to recognize, categorize, manage, and identify common radiographic pathologic conditions. Educators cannot rely on National Board scores and course grades to determine student clinical competency. More radiology clinical competency exercises that emphasize film interpretation need to be incorporated into content-based curricula.

Activin family members in the developing chick retina: expression patterns, protein distribution, and in vitro effects.

We have investigated whether the activin family of growth factors is involved in the regulation of retinal cell differentiation. Immunocytochemistry and in situ hybridization have shown that activin/inhibin subunits alpha, betaA, and betaB; receptors II and IIB; follistatin; and a follistatin-like gene are expressed in different regions of the chick embryo retina in developmentally regulated patterns. When tested in dissociated retinal cultures, activin did not appear to affect cell survival or proliferation, but it exerted marked inhibitory effects on the differentiation of photoreceptors, while stimulating the differentiation of nonphotoreceptor neurons; both effects were concentration-dependent and follistatin-sensitive. The results are consistent with the possibility that activin family members play significant roles in the regulation of retinal development.

Children's illness drawings and asthma symptom awareness.

This study examines the relationship between children's abilities to perceive their symptoms of asthma via several previously researched subjective and objective procedures compared with their performance on a standardized children's drawing task and scale criteria. Results indicated that girls verbalized significantly more emotions about their drawings and were better able to detect airflow changes in their small airways than boys. The Gabriels Asthma Perception Drawing Scales (GAPDS) is a promising clinical tool for assessing children's perceptions and emotions about asthma via nonverbal methods. Varying methods of measuring asthma symptom awareness are not highly correlated; thus, more than one methodology is appropriate for use with children.

A cauliflower mosaic virus promoter directs expression of kanamycin resistance in morphogenic transformed plant cells.

The promoter region of the CaMV inclusion body protein gene was modified for use in chimeric gene fusions. The modified promoter was used to construct a selectable marker for plant transformation based on the Tn 5 kanamycin resistance gene. This chimeric selectable marker was introduced into plant cells using oncogenic and deoncogenized strains of Agrobacterium tumefaciens. Both types of transformation produced kanamycin-resistant cell lines. The resistant cell lines derived from the deoncogenized strains were used to regenerate shoots. A second type of selection based on the ability of octopine synthase to detoxify aminoethyl cysteine was also used to select transformants in both oncogenic and nononcogenic transformation.

Microenvironmental regulation of visual pigment expression in the chick retina.

Visual pigment (VP) expression in the chick embryo retina was investigated in ovo, in dissociated and explant cultures, and in cDNAs from individual cells. While VP mRNA is not detectable by in situ hybridization until embryonic day (ED) 14-16 in ovo, analysis of VP expression by RT-PCR showed that VP messages are present in the retina as many as 7-10 days before they become detectable by in situ hybridization, and are also detected in other regions of the embryonic CNS. On the other hand, red opsin expression is markedly accelerated when cells are isolated from their intraocular microenvironment at ED 6, and placed in pigment epithelium-free dissociated or explant cultures. This acceleration occurs regardless of cell density, birth date, or serum presence in the medium, suggesting that many photoreceptors are already programmed to express red opsin on or before ED 6, and that microenvironmental inhibitory factors prevent implementation of this program until ED 14 in ovo. The selectivity of this phenomenon is suggested by the finding that other VPs are not observed by in situ hybridization in ED 6 cultures, although they are detectable in cultures of older retinas. Taken together, these findings suggest that red opsin expression may be constitutive for many developing photoreceptor cells in the chick.

Yunnanxane and its homologous esters from cell cultures of Taxus chinensis var. mairei.

From cell cultures of Taxus chinensis var. mairei, yunnanxane [2 alpha, 5 alpha, 10-beta triacetoxy-14 beta-(2'-methyl-3'-hydroxyl)-butyryloxy-4(20),11-taxadiene, [1], and four new homologous esters, 2 alpha, 5 alpha, 10 beta, 14 beta- tetra-acetoxy-4(20),11-taxadiene [2], 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-propionyloxy-4(20),11-taxadiene [3], 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-isobutyryloxy-4(20),11- taxadiene [4], and 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-(2'-methyl)-butyryloxy-4(20),11- taxadiene [5] have been isolated. Their structures were determined by spectroscopic methods.

New bioactive taxoids from cell cultures of Taxus baccata.

Four new taxoids were isolated from cell cultures of Taxus baccata. Their structures were elucidated by spectroscopic analyses. Two were the aglycones corresponding to previously isolated 7-O-xylosides of taxol C [1] and 10-deacetyltaxol C [2]. The third [3] had an N-methylated side-chain, while the fourth, named taxcultine [4], contained an n-propyl group on the side-chain. All four compounds actively promoted tubulin assembly. Taxol C [1] showed potent and selective cytotoxicity in the NCI human cell line screen.