A FORENSIC AND PHYLOGENETIC SURVEY OF CAULERPA SPECIES (CAULERPALES,CHLOROPHYTA) FROM THE FLORIDA COAST,LOCAL AQUARIUM SHOPS,AND E‐COMMERCE: ESTABLISHING A PROACTIVE BASELINE FOR EARLY DETECTION1

Baseline genotypes were established for 256 individuals of Caulerpa collected from 27 field locations in Florida (including the Keys), the Bahamas, US Virgin Islands, and Honduras, nearly doubling the number of available GenBank sequences. On the basis of sequences from the nuclear rDNA‐ITS 1+2 and the chloroplast tufA regions, the phylogeny of Caulerpa was reassessed and the presence of invasive strains was determined. Surveys in central Florida and southern California of >100 saltwater aquarium shops and 90 internet sites revealed that >50% sold Caulerpa. Of the 14 Caulerpa species encountered, Caulerpa racemosa was the most common, followed by Caulerpa sertularioides, Caulerpa prolifera, Caulerpa mexicana, and Caulerpa serrulata. None of the >180 field‐collected individuals (representing 13 species) was the invasive strain of Caulerpa taxifolia or C. racemosa. With one exception (a sample of C. racemosa from a shop in southern California belonged to the invasive Clade III strain), no invasive strains were found in saltwater aquarium stores in Florida or on any of the internet sites. Although these results are encouraging, we recommend a ban on the sale of all Caulerpa species (including “live rock”) because: morphological identification of Caulerpa species is unreliable (>12% misidentification rate) and invasive strains can only be identified by their aligned DNA sequences, and because the potential capacity for invasive behavior in other Caulerpa species is far from clear. The addition of the Florida region to the genetic data base for Caulerpa provides a valuable proactive resource for invasion biologists as well as researchers interested in the evolution and speciation of Caulerpa.     

Subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 assessed by immunohistochemistry.

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.     

ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting approximately 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis.     

The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 10⁵ PFU of virus, an E1A null mutant, pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant, pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU, pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.     

The impact of two gall-forming arthropods on the photosynthetic rates of their hosts

The impact of herbivores on host plant photosynthetic rates can range from negative to positive. While defoliation by chewing herbivores can result in increases in photosynthesis followed by compensatory growth, other herbivore guilds, such as mesophyll feeders which damage photosynthetic leaf tissues, almost always reduce photosynthetic rates. The impact of galling herbivores on host photosynthesis has rarely been examined, even though the limited tissue disruption and the strong metabolic sinks induced by gall-forming herbivores could potentially stimulate photosynthetic rates. I examined the hypothesis that gall-inducing herbivores could stimulate photosynthesis in neighboring leaves in response to increased sink-demand by the gall. To address this hypothesis, I measured photosynthetic rates of galled leaves or leaflets, neighboring ungalled leaves or leaflets, and ungalled leaves or leaflets on ungalled shoots on naturally growing Prunus serotina (wild cherry) and Rhus glabra (smooth sumac). The leaves of wild cherry were galled by an eriophyid mite, Phytoptus cerasicrumena; the leaves of smooth sumac by an aphid, Melaphis rhois. I found that both species reduced the photosynthetic rates of the leaves or leaflets they galled from 24 to 52% compared to ungalled leaves in ungalled areas of the plants. Contrary to my hypothesis, mite galls on wild cherry reduced photosynthesis of neighboring ungalled leaves within the same shoot by 24% compared to ungalled leaves on gall-free shoots. Aphid galls on sumac leaflets did not significantly alter the photosynthetic rates of neighboring leaflets relative to ungalled leaves on ungalled shoots. Although gall-formers would appear to have the potential to stimulate photosynthesis in the same manner as defoliating herbivores, i.e., by increasing sink demand relative to source supply, I found only negative impacts on photosynthesis. I suggest that sink competition for nutrients between developing leaves and growing gall tissue may account for the negative impacts of sink-inducing gallers on photosynthesis. Received: 17 October 1997 / Accepted: 2 February 1998     

FISH studies of the sperm of fathers of paternally derived cases of trisomy 21: no evidence for an increase in aneuploidy

Paternal nondisjunction accounts for approximately 5% of cases of trisomy 21. To test the hypothesis that, in some such cases, the fathers might be predisposed to meiotic nondisjunction, we utilized fluorescence in situ hybridization (FISH) to screen for aneuploidy in sperm. We analyzed sperm samples from ten males with a trisomy 21 offspring of paternal origin. Among these individuals, the overall frequency of disomy 21 was 0.15%, comparable to estimates of disomy 21 in the general male population. Furthermore, none of the ten fathers of trisomy 21 individuals had significantly elevated levels of disomic sperm. Thus, our results provide no evidence that the occurrence of a trisomy 21 conceptus of paternal origin imparts an increased risk of trisomy in subsequent pregnancies. Received: 9 September 1998 / Accepted: 30 September 1998     

Contribution of the allelicMtz 3 andMtz 4 allotype genes to the formation of individual rabbit serum α2-macroglobulin molecules

The contribution of the allelicMtz ³ andMtz ⁴ genes to the formation of individual rabbit serum α₂-macroglobulin (α₂M) molecules was examined by precipitation of α₂M from rabbits of known genotype with antiallotype antisera. The α₂M was isolated fromz ³z³ andz ⁴z⁴ homozygous andz ³z⁴ heterozygous rabbits, iodinated with I¹²⁵ and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α₂M or α₂M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α₂M. Nearly all α₂M molecules have z3 or z4 antigenic determinants; approximately 50% of α₂M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60% of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known allelic blood protein systems of rabbits, allelic exclusion does not occur in α₂M molecules of heterozygotes; rather, hybrid molecules are formed. Presented in part at the Fifty-fourth and Fifty-fifth Annual Meetings of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 12–17, 1970, and Chicago, Illinois, April 12–17, 1971. This investigation was supported in part by U.S. Public Health Service Grants AI-09241 and AI-07043. B.H.B. performed this investigation in partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College; he is supported by a postdoctoral fellowship from the Schweppe Foundation. K.L.K. is the recipient of U.S. Public Health Service Research Career Development Award AI-28687.     

Glycerylphosphorylcholine phosphodiesterase in rat liver. Subcellular distribution and localization in plasma membranes

1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5'-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5'-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a K(m) for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe(3+)).