CELL DIVISION AND THE ROLE OF THE PRIMARY WALL IN THE FILAMENTOUS DESMID ONYCHONEMA LAEVE (CHLOROPHYTA)1

Cell division and the role of the primary wall in filament formation in the desmid Onychonema laeve Nordst. were investigated by transmission and scanning electron microscopy. In addition, sequential chemical extractions and enzyme treatments were performed, on cell walls of intact filaments. Interphase cells are deeply constricted, consisting of two semicells, each elliptical in front view and circular in side view. In addition to two short lateral spines, each semicell has two apical processes that originate on opposite sides at an angle of about 15° from the central axis and overlap the adjacent cell. Division is initiated as in other desmids by a slight separation of the semicells and development of a girdle of primary wall material at the isthmus. In O. laeve the girdle of primary wall expands to form a spherical vesicle (termed a division vesicle) between the separating semicells. Nuclear division and septum formation occur in this vesicle when it is nearly the full diameter of the filament. Morphogenesis of the apical processes begins with completion of the septum, before the secondary wall appears. At maturity each apical process is surrounded by a thick layer of both secondary and primary wall, except that its capitate tip protrudes through the shroud of primary wall. Sequential treatment with hot ammonium oxalate, 4% NaOH, 17.5% NaOH and 10% chromic acid or various enzyme solutions did not cause filament breakage. SEM and TEM views of O. laeve after these treatments show intact secondary walls and intact primary wall material covering and connecting the apical processes of adjacent cells. It is the persistence of the primary wall between cells and around the apical processes that maintains the long, unbranched filamentous morphology of Onychonema laeve.     

Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Formation of domoic acid and fatty acids inPseudonitzschia pungens f.multiseries with scale of culture

Pseudonitzschia pungens f.multiseries was cultured in 20-L polycarbonate carboys, 350-L fibreglass columns and 500-L plastic bags to determine the effects of medium enrichment and scale of culture on cell yield, production of cellular domoic acid and formation of fatty acids, particularly the potential tracer acid 16:4n-1. Cell concentrations were highest in seawater enriched with stock levels of nitrate and phosphate, but with double the stock level of silicate, at all scales of culture. Cellular toxin in 20, 350 and 500-L cultures averaged 0.32, 0.04 and 2.56 pg cell^-1 and was independent of medium used. The order of magnitude difference in levels of cellular toxin was considered to reflect the varying levels of irradiance within the culture vessels. Support was given to this by the significant difference in content of total cellular fatty acids, due principally to the algal storage acid 16: 1n-7, which is known to be influenced by irradiance. Levels of cellular domoic acid correlated significantly with total fatty acids in 350 and 500-L cultures. Bag cultures producing significantly higher levels of cellular domoic acid provided lower relative proportions of 16:4n-1, which limited its use as a tracer for food-web studies.     

Studies of sibling Drosophila species from Laguna Verde, Veracruz, Mexico: I. Species frequencies, viability, desiccation resistance, and vagility

The sibling species Drosophila melanogaster and D. simulans were collected at Laguna Verde, Veracruz, Mexico. D. melanogaster was found in significantly greater frequency than was D. simulans. Ten isofemale lines of each species were tested for egg to adult viability, desiccation resistance, and vagility. D. melanogaster surpassed D. simulans in all three characteristics. The findings are discussed with reference to the climatic conditions at Laguna Verde and the expected effect of such an environment on the relative frequencies of these species. The dichotomous results in regard to desiccation resistance and vagility that were observed between recently collected D. melanogaster and the Oregon-R laboratory stock of that species are also discussed.     

K+-independent effects of valinomycin in photosynthetic systems

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K⁺. NH₄Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH₄Cl plus valinomycin but not by either alone. K⁺ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH₄Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg²⁺-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H⁺/e^– ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.     

Efficacy of a Vaccine Formula against Tuberculosis in Cattle

“Test-and-slaughter” has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10⁶ CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10⁵ CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.     

3,5-Bis(benzylidene)-4-piperidones and related N-acyl analogs: A novel cluster of antimalarials targeting the liver stage of Plasmodium falciparum

Drug resistance is a major challenge in antimalarial chemotherapy. In addition, a complete cure of malaria requires intervention at various stages in the development of the parasite within the host. There are only a few antimalarials that target the liver stage of the Plasmodium species which is an essential part of the life cycle of the malarial parasite. We report a series of antimalarial 3,5-bis(benzylidene)-4-piperidones and related N-acyl analogs 1–5, a number of which exhibit potent in vitro growth-inhibiting properties towards drug-sensitive D6 and drug-resistant C235 strains of Plasmodium falciparum as well as inhibiting the liver stage development of the malarial life cycle. The compounds 2b (IC₅₀: 165 ng/mL), 3b (IC₅₀: 186 ng/mL), 5c (IC₅₀: 159 ng/mL) and 5d (IC₅₀: 93.5 ng/mL) emerged as lead molecules that inhibit liver stage Plasmodium berghei and are significantly more potent than chloroquine (IC₅₀: >2000 ng/mL) and mefloquine (IC₅₀: >2000 ng/mL) in this screen. All the compounds that showed potent inhibitory activity against the P. berghei liver stage were nontoxic to human HepG2 liver cells (IC₅₀: >2000 ng/mL). The compounds 5a and 5b exhibit comparable metabolic stability as chloroquine and mefloquine in human plasma and the most potent compound 5d demonstrated suitable permeability characteristics using the MDCK monolayer. These results emphasize the value of 3,5-bis(benzylidene)-4-piperidones as novel antimalarials for further drug development.     

Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca²⁺ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca²⁺-binding EF hands. Ca²⁺ stores are refilled through a process called store-operated Ca²⁺ entry (SOCE). Upon Ca²⁺-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca²⁺ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca²⁺ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca²⁺ level in TAM cells and a dysregulation of intracellular Ca²⁺ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.     

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4⁺ and CD8⁺ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.