Dynamic visualization of thrombopoiesis within bone marrow

Platelets are generated from megakaryocytes (MKs) in mammalian bone marrow (BM) by mechanisms that remain poorly understood. Here we describe the use of multiphoton intravital microscopy in intact BM to visualize platelet generation in mice. MKs were observed as sessile cells that extended dynamic proplatelet-like protrusions into microvessels. These intravascular extensions appeared to be sheared from their transendothelial stems by flowing blood, resulting in the appearance of proplatelets in peripheral blood. In vitro, proplatelet production from differentiating MKs was enhanced by fluid shear. These results confirm the concept of proplatelet formation in vivo and are consistent with the possibility that blood flow-induced hydrodynamic shear stress is a biophysical determinant of thrombopoiesis.     

Degradation of insecticide lindane (γ‐HCH) by white‐rot fungi Cyathus bulleri and Phanerochaete sordida

An Erratum for this article has been published in Pest Management Science 56(5) 493 (2000). The degradation of the insecticide lindane (γ‐hexachlorocyclohexane, γ‐HCH) by two white‐rot fungi, Cyathus bulleri and Phanerochaete sordida, was studied. C bulleri degraded lindane more efficiently than P sordida. Two degradative intermediates identified in P sordida culture were tetrachlorocyclohexene and tetrachlorocyclohexanol. However, tetrachlorocyclohexanol was the sole degradation product detected in cultures of C bulleri. The presence of lindane only inside the mycelial cells of both fungi eliminated any role of intracellular enzymes during initial steps of its degradation. The insecticide at 0.27 µM showed no adverse effect on fungal growth. © 2000 Society of Chemical Industry     

Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

Honey Bee (Hymenoptera: Apidae) Nursing Responses to Cuticular Cues Emanating from Short-term Changes in Larval Rearing Environment

Honey bee larvae are dependent on the social structure of colony for their provisioning and survival. With thousands of larvae being managed collectively by groups of foragers (collecting food resources) and nurse bees (processing food and provisioning larvae), coordination of colony efforts in rearing brood depends on multiple dynamic cues of larval presence and needs. Much of these cues appear to be chemical, with larvae producing multiple pheromones, major being brood ester pheromone (BEP; nonvolatile blend of fatty acid esters) that elicits both short-term releaser effects and long-term primer effects. While BEP can affect colony food collection and processing with the signaling of larval presence, it is unclear if BEP signals individual larval needs. To understand this aspect, in a series of experiments we manipulated larval feeding environment by depriving larvae from adult bee contact for 4-h period and examined (1) nurse bee interactions with contact-deprived and nondeprived larvae and larval extracts; (2) forager bee responses to contact-deprived and nondeprived larval extracts. We also characterized BEP of contact-deprived and nondeprived larvae. We found that nurse honey bees tend to aggregate more over contact-deprived larvae when compared with nondeprived larvae, but that these effects were not found in response to whole hexane extracts. Our analytical results suggest that BEP components changed in both quantity and quality over short period of contact deprivation. These changes affected foraging behavior, but did not appear to directly affect nursing behavior, suggesting that different chemical cues are involved in regulating nursing effort to individual larvae.     

Low level of polymorphism detected by SSR probes in bread wheat

In-gel hybridization patterns were studied in a set of nine diverse bread wheat ( Triticum aestivum L. em. Thell) genotypes using 23 simple sequence repeat (SSR) probes in combination with 14 different restriction enzymes. Multilocus fingerprints due to SSR probes, shown earlier to be characteristic of a majority of plant genomes, were not obtained and only a very low level of polymorphism was detected when using as many as 142 probe-enzyme combinations. The hybridization of a prominent solitary high molecular weight fragment (> 23 kb) with a number of SSR probes suggested the presence of these SSRs (microsatellites) within the long stretches of repeated DNA sequences. This indicates that the genome of bread wheat differs from that of other plants in the organization and distribution of SSRs and that SSR probes detect very little polymorphism.     

Genetic Diversity in Colletotrichum gloeosporioides from Stylosanthes spp. at Centers of Origin and Utilization

ABSTRACT Using molecular markers, this work compares the genetic diversity in Colletotrichum gloeosporioides infecting species of the tropical forage legume Stylosanthes at the center of origin in Brazil and Colombia with that of Australia, China, and India, where Stylosanthes spp. have been introduced for commercial use. There was extensive diversity in the pathogen population from Brazil, Colombia, China, and India. The Australian pathogen population was least diverse probably due to its geographical isolation and effective quarantine. The extensive diversity in China and India means that threats from exotic pathogen races to Stylosanthes pastures can potentially come from countries outside the South American center of origin. In Brazil and India, both with native Stylosanthes populations, a high level of genetic differentiation in the pathogen population was associated with sites where native or naturalized host population was widely distributed. There was limited genetic diversity at germplasm evaluation sites, with a large proportion of isolates having identical haplotypes. This contrasts recent pathogenicity results for 78 of the Brazilian isolates that show hot spots of complex races are more common around research stations where host germplasm are tested, but few are found at sites containing wild host populations. For a pathogen in which the same races arise convergently from different genetic backgrounds, this study highlights the importance of using both virulence and selectively neutral markers to understand pathogen population structure.     

Survival and development of spotted bollworm, <Emphasis Type="Italic">Earias vittella</Emphasis> (Fabricius) (Lepidoptera: Nolidae) on different transgenic <Emphasis Type="Italic">Bt</Emphasis> and isogenic non-<Emphasis Type="Italic">Bt</Emphasis> cotton genotypes

Four Bt cotton hybrids, each with one of four different events, viz., MRC 6301 Bt (cry1Ac gene), JKCH 1947 Bt (modified cry1Ac gene), NCEH 6R Bt (fusion cry1Ac/cry1Ab gene) and MRC 7017 Bollgard II (cry1Ac and cry2Ab genes) were compared for survival and development of Earias vittella (Fabricius) along with their isogenic non-Bt genotypes. None of the neonates were able to complete the larval period and reach pupal stage on squares of 90, 120 and 150 days old crop of all Bt hybrids. Likewise, on bolls also, zero per cent larval survival was observed in all Bt hybrids except JKCH 1947 Bt where 0.67 per cent larvae could manage to reach pre-pupal stage at 120 and 150 days old crop but failed to form cocoon and enter pupal stage. The surviving larva took more development time (3.7 to 5.4 days) as compared to larvae fed on bolls of JKCH 1947 non-Bt. The average survival period (ASP) of larvae was in order of 150 > 120 > 90 days old crop among the crop ages; JKCH 1947 Bt > MRC 6301 Bt > NCEH 6 R Bt > MRC 7017 Bollgard II among Bt hybrids; and bolls > squares between fruiting bodies. However, reverse was true for speed index of toxic effect. The concentration of Cry toxin varied significantly in squares and bolls and also among the crop ages. The amount of Cry toxin in squares and bolls had significant negative correlation with ASP of the E. vittella larvae.