Recent Mediterranean fruit fly (Diptera: Tephritidae) infestations in Florida--a genetic perspective

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study infestations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) in Florida in 1997 and 1998. A total of 132 flies collected in monitoring traps or as larvae removed from fruit were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. All of the flies sampled in Florida in 1997 displayed the mitochondrial AAB haplotype and represent a novel introduction of Mediterranean fruit flies into the state. All flies collected in central Florida in 1998 also displayed the AAB haplotype. Microsatellite analysis of these specimens from 1998 detected only alleles that were present in 1997. These results strongly indicate that the 1998 Florida outbreaks were derived from the Florida populations from the previous year. According to our analyses, the Mediterranean region is the most likely source for the 1997 Florida infestation. Flies from a small outbreak in Miami Springs, Dade County, FL, early in 1998 had a different mtDNA haplotype, characterized by the AAC restriction pattern. Microsatellites of these specimens showed significant differences in their allelic distribution from AAB flies, indicating an origin from a separate source population. South America is the most likely source for the Miami Springs flies.     

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: Growth characteristics,cytolytic activity,and cell phenotype

Summary Outgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard IL-2-only method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 10²³ TIL from as few as 2 × 10⁵ cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8⁺ and CD4⁺ cells, with few CD16⁺ or NKH-1⁺. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4⁺. Further analysis of these CD4⁺ TIL indicated that all were of the helper-inducer class, 4B4⁺ and 2H4^–. Concurrent with the decline in CD8⁺ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8⁺, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.This work was supported by a grant from the Dunn Foundation, Houston, Texas     

Shift in the spatial and temporal distribution of Aedes taeniorhynchus following environmental and local developments in St. Johns County,Florida

The Anastasia Mosquito Control District (AMCD) of St. Johns County (SJC), St. Augustine, Florida, USA, was formed in 1948 to cover the 27 km² of Anastasia Island and control the black salt marsh mosquito, Aedes taeniorhynchus (Wiedemann). Today AMCD covers the entirety of SJC (1588 km²) and Ae. taeniorhynchus is still the most abundant mosquito species in the county. Here we present the findings from 16 years’ worth of surveillance records of AMCD mosquito populations in conjunction with annual land-use land-cover (LULC) change and climate data to better understand how environmental factors have impacted SJC Ae. taeniorhynchus populations in recent history. The statistical regression and geospatial analyses demonstrated the presence of spatial and temporal clusters of Ae. taeniorhynchus populations in terms of abundance and distribution. Additionally, Ae. taeniorhynchus abundance and distribution were significantly influenced by the annual changes of LULC and climate variables. The linear regression analysis using standard least square and corrected Akaike Information Criterion revealed a migration of mangrove swamps and saltwater marshes that corresponded to a southern shift in the spatial–temporal distribution of Ae. taeniorhynchus communities. This was confirmed by the significant change in LULC characteristics between three representative years (2004, 2009, 2014) and the redistribution of Ae. taeniorhynchus abundances represented by Moran’s I index values. The annual values of four climate variables (average and minimum temperature, mean dew point, and maximum vapor pressure deficit) and three LULC types (mangrove swamps, saltwater pools within saltmarshes, and upland nonforested) significantly predicted annual abundance and redistribution of Ae. taeniorhynchus.

     

VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence.

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.     

Mycolic acids. A reinvestigation.

Mycolic acids derived from the cell walls of Mycobacterium bovis BCG, Mycobacterium bovis Bovinus I, Mycobacterium smegmatis, and Mycobacterium tuberculosis H37Rv have been fractionated as their p-bromophenacyl esters by a two-step high performance liquid chromatographic procedure: 1) adsorption chromatography on 10-micrometer particle size silica gel, and 2) reverse phase partition chromatography on a 10-micrometer particle size support containing a C18 bonded phase. This procedure has resulted in the isolation of approximately 24 mycolic acids from each bacterium (very likely homologs of various mycolate types) instead of the two to four that have previously been described. The implication of these results on the previously determined structures of these fatty acids is discussed.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Interaction of the aldolase and the membrane of human erythrocytes.

Up to 80% of cellular aldolase (EC 4.1.2.13) was retained in the membrane fraction isolated following hemolysis of human erythrocytes under appropriate conditions. Binding was reversed by increasing the pH and ionic strength. Millimolar levels of the substrate, fructose 1,6-bisphosphate, selectively eluted aldolase from the membrane, while related metabolites did not. Using the membrane as a high affinity adsorbant, electrophoretically pure aldolase of high specific activity was prepared in high yield. The reassociation of pure aldolase and membranes was characterized. The sole site of human erythrocyte aldolase binding was shown to be the cytoplasmic surface domain of band 3, the predominant membrane-spanning polypeptide. One aldolase molecule was bound per band 3 polypeptide. Upon binding to either whole membranes, solubilized band 3, or proteolytic fragments from the cytoplasmic surface pole of band 3, aldolase underwent a profound loss of catalytic activity, reversed by raising the substrate concentration.     

Fine structure of the band 3 protein in human red cell membranes: Freeze-fracture studies

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30–50 Å in diameter, the “ES particles.” The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisonally identified as the 40,000-dalton inner-surface domain of band 3.     

Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.