Critical stages in the development of Plasmodium in mosquitoes

One tool for the control of malaria that may become available to future generations of public health workers is the introduction of genes into the Anopheline vector populations that will render the mosquitoes refractory to Plasmodium. Insights from basic research that could transform this idea into a technical reality are presently lacking. In this review, Alon Warburg and Louis Miller focus on one crucial area of research: the identification of potentially vulnerable points in the developmental cycle of Plasmodium in mosquitoes.     

Transmission and scanning EM-immunogold labeling of Leishmania major lipophosphoglycan in the sandfly Phlebotomus papatasi

Previous studies using immunostaining and light microscopy demonstrated expression of Leishmania major lipophosphoglycan (LPG) on parasites developing in the sandfly gut from 2 days post infection. By days 4 to 7 post infection, there appeared to be large amounts of parasite-free LPG deposited on/in the microvilli and epithelial cells lining the thoracic midgut, while forward migration of parasites and the morphological changes which accompany metacyclogenesis were associated with developmental modification of the LPG molecules. Studies presented here examine this process with much greater precision using electron microscopy and immunogold labeling techniques to study the different developmental forms (nectomonads, haptomonads, paramastigotes, and metacyclics) of promastigotes in the sandfly gut. Results obtained using LPG-specific monoclonal antibodies (WIC79.3, 45D3 and the metacyclic-specific 3F12) show (1) gold labeling over the cell surface, within the flagellar pocket, and extending along the entire length of the flagellum of electron-dense nectomonads observed in the abdominal and thoracic midgut regions on days 4 and 7 post infection, and of electron-lucid haptomonads in the foregut, (2) dense labeling around the flagellar tips, by which nectomonad forms bind to the midgut microvilli, but not on the microvilli themselves or within the epithelial cells lining the midgut, (3) significant metacyclic-specific (3F12) labeling on nectomonad forms in the lumen of the midgut and attached to the microvilli, and (4) dense labeling on the cell surface of electron-lucid paramastigotes in the esophagus and in the filamentous matrix surrounding paramastigote and metacyclic forms in the esophagus and pharynx. These results are discussed in the light of the proposed roles for LPG in parasite attachment to, and survival in, the sandfly gut.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Oosorption and oocyte loss in a terrestrial isopod under stressful conditions

An increased number of resorbed oocytes was observed in ovaries of terrestrial isopods which were kept under different experimental temperature and photophase regimes compared with those observed in the natural population. Regardless of the nature of the stimulus: high or low temperature or long and short photophases, the female always responded through oosorption. In an iteroparous species such as Porcellio ficulneus B.-L., recruitment of resources for future utilization could be its main response to adverse ambient conditions.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus

Facioscapulohumeral muscular dystrophy (FSHD) is a relatively common autosomal dominant neuromuscular disorder. The gene for FSHD has recently been assigned to chromosome 4q35. Although abnormal mitochondrial and biochemical changes have been observed in FSHD, the molecular defect is unknown. In addition to the FSHD gene, the human muscle adenine nucleotide translocator gene (ANT1) is located on chromosome 4. Interestingly, biochemical studies recently showed a possible defect of ANT1. In order to evaluate the potential role of ANT1 in the etiology of FSHD, the human ANT1 gene was isolated by cosmid cloning and localized to 4q35, in the region containing the FSHD gene. However, in situ hybridization and physical mapping of somatic cell hybrids localized the ANT1 gene proximal to the FSHD gene. In addition, a polymorphic CA-repeat 5 kb upsstream of the ANT1 gene was used as a marker in FSHD and Centre d'Etude du Polymorphisme Humain families to perform linkage analysis. These data together exclude ANT1 as the primary candidate gene for FSHD. The most likely order of the loci on chromosome 4q35 is cen-ANT1-D4S171-F11-D4S187-D4S163-D4S139-FSHD-tel.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.