Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

Regulation of Trichodiene Synthase in Fusarium sporotrichioides and Gibberella pulicaris (Fusarium sambucinum)

The regulation of trichodiene synthase (TS) and its relationship to trichothecene biosynthesis was investigated in Fusarium sporotrichioides NRRL 3299 and Gibberella pulicaris R-6380. Cultures were analyzed for the presence of TS activity, trichothecenes, and immunodetectable TS polypeptide over a time period of 144 h. Enzyme activity increased from barely detectable to maximum levels over a period of 3 h for F. sporotrichioides, while in G. pulicaris, a steady increase was observed over 144 h. Increases in TS activity of 50-fold for F. sporotrichioides and 10-fold for G. pulicaris R-6380 preceded by several hours the detection of trichothecenes. Immunoblot analysis employing polyclonal serum specific for the enzyme from F. sporotrichioides showed that increases in the levels of TS polypeptide corresponded to the observed changes in enzyme activity for both organisms. These data indicate that the regulation of TS activity is accomplished through increases in its cellular concentration and that TS may serve as a useful indicator of trichothecene biosynthetic activity.     

REPRODUCTIVE SEASONALITY OF WESTERN ATLANTIC BOTTLENOSE DOLPHINS OFF NORTH CAROLINA, U.S.A.

We describe reproductive seasonality of bottlenose dolphins in North Carolina (NC), U.S.A., using strandings data from the entire coast of NC and sighting data from Beaufort, NC and by estimating dates of birth of known females. We found a strong peak of neonate strandings in the spring (April-May), and low levels of neonate strandings in the fall and winter. The distribution of neonate strandings was significantly different from a uniform distribution ( P < 0.001, K = 3.8). We found a unimodal distribution of 282 sightings of neonates with a diffuse peak in the summer. The temporal distribution of sightings of neonates departed significantly from a uniform distribution (P < 0.001, K = 5.1). Estimated birth dates of neonates from known females occurred in May ( n = 6) and June ( n = 4), with a single fall birth. These methods shed light on bottlenose reproductive patterns and underscore the value of using information from multiple types of data. Clarification of bottlenose dolphin reproductive patterns, such as the seasonality of birth, may enhance our understanding of the population structure of this species in the mid-Atlantic region.     

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein

In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus. Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process. We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently. In this study, we performed a mutational analysis of the roles played by A. tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A. tumefaciens carrying pTF-FC2. We show that MobA+/VirD2+ and MobA+/VirD2– strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them. However, the MobA–/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains. This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro. We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2.     

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.