"Pesta": New Granular Formulations for Steinernema carpocapsae

"Pesta," a new granular product for use with entrapped biocontrol agents, is based on a cohesive dough made of wheat flour, fillers, and other additives. Infective juveniles of the entomopathogen Steinernema carpocapsae strain All incorporated in Pesta granules emerged when the granules were softened by immersion in water. These granules may be useful for the biocontrol of insect pests in the soil. Storage temperature had the greatest effect on recovery of nematodes, followed by the moisture content of the granules. Recovery of nematodes was the same among the formulations tested and was unaffected by storage in nitrogen. Nematode recovery after storage at 21 C decreased to zero after 3-6 weeks. Storage of samples at 4 C and with a high moisture content (19.9-23.1%) greatly improved nematode viability.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Human immunodeficiency virus type 1 bound to B cells: relationship to virus replicating in CD4+ T cells and circulating in plasma

Human immunodeficiency virus type 1 (HIV-1) virions bind to B cells in the peripheral blood and lymph nodes through interactions between CD21 on B cells and complement-complexed virions. B-cell-bound virions have been shown to be highly infectious, suggesting a unique mode of HIV-1 dissemination by B cells circulating between peripheral blood and lymphoid tissues. In order to investigate the relationship between B-cell-bound HIV-1 and viruses found in CD4+ T cells and in plasma, we examined the genetic relationships of HIV-1 found in the blood and lymph nodes of chronically infected patients with heteroduplex mobility and tracking assays and DNA sequence analysis. In samples from 13 of 15 patients examined, HIV-1 variants in peripheral blood-derived B cells were closely related to virus in CD4+ T cells and more divergent from virus in plasma. In samples from five chronically viremic patients for whom analyses were extended to include lymph node-derived HIV-1 isolates, B-cell-associated HIV-1 and CD4+-T-cell-associated HIV-1 in the lymph nodes were equivalent in their divergence from virus in peripheral blood-derived B cells and generally more distantly related to virus in peripheral blood-derived CD4+ T cells. These results indicates virologic cross talk between B cells and CD4+ T cells within the microenvironment of lymphoid tissues and, to a lesser extent, between cells in lymph nodes and the peripheral blood. These findings also indicate that most of the virus in plasma originates from cells other than CD4(+) T cells in the peripheral blood and lymph nodes.     

Selection for human immunodeficiency virus type 1 recombinants in a patient with rapid progression to AIDS

Although human immunodeficiency virus type 1 (HIV-1) recombinants have been found with high frequency, little is known about the forces that select for these viruses or their importance to pathogenesis. Here we document the emergence and dynamics of 11 distinct HIV-1 recombinants in a man who was infected with two subtype B HIV-1 strains and progressed rapidly to AIDS without developing substantial cellular or humoral immune responses. Although numerous frequency oscillations were observed, a single recombinant lineage eventually came to dominate the population. Numerical simulations indicate that the successive recombinant forms displaced each other too rapidly to be explained by any simple model of random genetic drift or sampling variation. All of the recombinants, including several resulting from independent recombination events, possessed the same sequence motif in the V3 loop, suggesting intense selection on this segment of the viral envelope protein. The outgrowth of the predominant V3 loop recombinants was not, however, associated with changes in coreceptor utilization. The final variant was instead notable for having lost 3 of 14 potential glycosylation sites. We also observed high ratios of synonymous-to-nonsynonymous nucleotide changes-suggestive of purifying selection-in all viral populations, with particularly high ratios in newly arising recombinants. Our study, therefore, illustrates the unusual and important patterns of viral adaptation that can occur in a patient with weak immune responses. Although it is hard to tease apart cause and effect in a single patient, the correlation with disease progression in this patient suggests that recombination between divergent viruses, with its ability to create chimeras with increased fitness, can accelerate progression to AIDS.     

Evolution of intrahost HIV-1 genetic diversity during chronic infection

HIV-1 is one of the fastest evolving entities known. Given that census population sizes of HIV-1 within individuals are much greater than the inverse mutation rate, every possible single point mutation in the viral genome occurs each generation. This enormous capability to generate genetic variation allows for escape from immune surveillance and antiviral therapy. However, compared to this potential, populations of HIV-1 within individuals exhibit little genetic variation. This discrepancy between the known mutation rate of HIV-1 and the average level of genetic variation in the env gene observed in vivo is reflected in comparisons of the actual numbers of productively infected cells, estimated as 10(7), and the effective population size, estimated as 10(3). Using approximate Bayesian computation, we evaluated several hypotheses based on a variety of selective and demographic processes to explain the low effective population size of HIV-1. Of the models we examined, the metapopulation model, in which HIV-1 evolves within an individual as a large collection of small subpopulations subject to frequent migration, extinction, and recolonization, was most consistent with the observed levels of genetic variation and the average frequencies of those variants. The metapopulation model links previous studies of viral dynamics and population genetics.     

Importance and detection of virus reservoirs and compartments of HIV infection

Current therapies for treating HIV-1 infection are capable of suppressing virus load in blood to undetectable levels, and result in marked clinical improvement. Despite this suppression, HIV-1 infection persists and virus load quickly rebounds when therapy is interrupted. The origin of the rebounding virus is unknown, but is thought to result from continuing viral replication in anatomic or cellular compartments, and the release of virus from latent infection in reservoirs.     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.