Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter

Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.     

Constructing more informative plant–pollinator networks: visitation and pollen deposition networks in a heathland plant community

Interaction networks are widely used as tools to understand plant–pollinator communities, and to examine potential threats to plant diversity and food security if the ecosystem service provided by pollinating animals declines. However, most networks to date are based on recording visits to flowers, rather than recording clearly defined effective pollination events. Here we provide the first networks that explicitly incorporate measures of pollinator effectiveness (PE) from pollen deposition on stigmas per visit, and pollinator importance (PI) as the product of PE and visit frequency. These more informative networks, here produced for a low diversity heathland habitat, reveal that plant–pollinator interactions are more specialized than shown in most previous studies. At the studied site, the specialization index was lower for the visitation network than the PE network, which was in turn lower than for the PI network. Our study shows that collecting PE data is feasible for community-level studies in low diversity communities and that including information about PE can change the structure of interaction networks. This could have important consequences for our understanding of threats to pollination systems.     

Space use and genetic structure do not maintain color polymorphism in a species with alternative behavioral strategies

Space use including territoriality and spatial arrangement within a population can reveal important information on the nature, dynamics, and evolutionary maintenance of alternative strategies in color polymorphic species. Despite the prevalence of color polymorphic species as model systems in evolutionary biology, the interaction between space use and genetic structuring of morphs within populations has rarely been examined. Here, we assess the spatial and genetic structure of male throat color morphs within a population of the tawny dragon lizard, Ctenophorus decresii. Male color morphs do not differ in morphology but differ in aggressive and antipredator behaviors as well as androgen levels. Despite these behavioral and endocrine differences, we find that color morphs do not differ in territory size, with their spatial arrangement being essentially random with respect to each other. There were no differences in genetic diversity or relatedness between morphs; however, there was significant, albeit weak, genetic differentiation between morphs, which was unrelated to geographic distance between individuals. Our results indicate potential weak barriers to gene flow between some morphs, potentially due to nonrandom pre‐ or postcopulatory mate choice or postzygotic genetic incompatibilities. However, space use, spatial structure, and nonrandom mating do not appear to be primary mechanisms maintaining color polymorphism in this system, highlighting the complexity and variation in alternative strategies associated with color polymorphism.     

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.     

Site-selective 8-chloroadenosine 3',5'-cyclic monophosphate inhibits transformation and transforming growth factor alpha production in Ki-ras-transformed rat fibroblasts

A site-selective cAMP analog, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), was demonstrated to be a potent inhibitor of both the monolayer and soft agar growth of normal rat kidney (NRK) fibroblasts that had been transformed with the v-Ki-ras oncogene or treated with transforming growth factor alpha (TGF alpha). The growth inhibition was dose dependent and reversible and was accompanied by reversion of the transformed phenotype, suppression of TGF alpha production, and a decrease in p21 ras protein levels. These effects of 8-Cl-cAMP were linked to the cAMP analog's selective modulation of the type I and type II cAMP-dependent protein kinase regulatory subunits, RI and RII, present in Ki-ras-transformed and TGF alpha-treated NRK cells.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Crypt fission and crypt number in the small and large bowel of postnatal rats

The aim of this investigation was to study crypt fission, a process which may be instrumental in regulating crypt number in the intestine. Young Holtzman rats were killed at various times after parturition and samples of the small intestine and colon were removed and processed. A microdissection technique was used to separate crypts from other structures. Crypts were scored as normal or fissioning. The percentage of crypts in fission (PCF) reached peak values of 25% and 52% in the small bowel and colon, respectively, at 21 days post-parturition. From this time onward, the PCF dropped until the adult value of approximately 7% was reached in each site. During this same period, the number of crypts increased from 1.9 X 10(6) to 3.3 X 10(6) in the small bowel and 2.2 X 10(5) to 6.5 X 10(5) in the colon. Thus an inverse relationship between the percentage of crypts in fission and crypt number was found. Distribution of fissure heights in fissioning crypts did not change as the animal aged. The majority of the fissures were found in the lower 1/4 of the fissioning crypts. This suggests that as soon as the fissure extends beyond the stem cell zone, division into two crypts soon occurs.     

The Latex Serologic Test for Syphilis—A New Screening Procedure

A new macroscopic screening test for syphilis, the Latex-sts test, is extraordinarily simple. After inactivation of the patient''s serum for 30 minutes at 56°C the test is performed by mixing the patient''s serum with latex particles coated with cardiolipin and a protein fraction obtained from the non-pathogenic Reiter strain of Treponema pallidum. Two to three minutes after mixing, the result of the test is observed on a ringed serologic plate. The sensitivity, specificity and reproducibility of the new test are equivalent to those of the qualitative Venereal Disease Research Laboratory tube test. The advantages of the Latex-sts are that it can be done in a short time, it is simple and it requires a minimum of laboratory equipment. The coated latex particles are stable for 12 months.     

Inheritance of resistance to blackmold (Alternaria alternata (Fr.) Keissler) in two interspecific crosses of tomato (Lycopersicon esculentum ×L. cheesmanii f.typicum)

Inheritance of resistance to blackmold, a disease of ripe tomato fruit caused byAlternaria alternata, was studied in two interspecific crosses. The parents, F₁ and F₂ generations of a cross between the susceptibleLycopersicon esculentum Mill. cultivar Hunt 100 and the resistantL. Cheesmanii f.typicum Riley accession LA 422, and the parents, F₁, F₂, F₃, and BC₁ P₂ generations of a cross between the susceptibleL. Esculentum cv. VF 145B-7879 and LA 422 were evaluated. The following disease evaluation traits were used: symptom rating (a symptom severity rating based on visual evaluation of lesions), diseased fruit (the number of diseased fruits divided by the total number of fruit scored), and lesion size (a function derived from the actual lesion diameter). Generation means analysis was used to determine gene action. The data of the Hunt 100 × LA 422 cross fit an additive-dominance model for all three traits. The VF 145B-7879 × LA 422 cross data best fit a model that included the additive × additive and additive × dominance interaction components for the trait diseased fruit, whereas higher-order epistatic models would have to be invoked to fit the data for the traits symptom rating and lesion size. A minimum of one gene segregated for all three traits. Broad-sense heritability estimates ranged from 0.09 to 0.16 for all three traits, indicating that selection for improved resistance to blackmold will require selection on a family performance basis.