A rapid method for preparing insect microsomes

The 1000 g supernatant of a tissue homogenate is layered on top of a small (less than 5 ml) sucrose density gradient and centrifuged for 20 min at very high centrifugal forces in a vertical rotor. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centrifugation method. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.     

Wolbachia from the planthopper Laodelphax striatellus establishes a robust, persistent, streptomycin-resistant infection in clonal mosquito cells

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales: Anaplasmataceae), distorts reproduction of its arthropod hosts to facilitate invasion of naïve populations. This property makes Wolbachia an attractive “gene drive” agent with potential applications in the control of insect vector populations. Genetic manipulation of Wolbachia will require in vitro systems for its propagation, genetic modification, amplification, and introduction into target insects. Here we show that Wolbachia from the planthopper, Laodelphax striatellus, establishes a robust infection in clonal C7-10 Aedes albopictus mosquito cells. Infected cells, designated C/wStr, expressed radiolabeled proteins that were enriched in cells grown in the absence of antibiotics that inhibit Wolbachia, relative to cultures grown in medium containing tetracycline and rifampicin. Using mass spectrometry, we verified that tryptic peptides from an upregulated 24 kDa band predominantly represented proteins encoded by the Wolbachia genome, including the outer surface protein, Wsp. We further showed that resistance of Wolbachia to streptomycin is associated with a K42R mutation in Wolbachia ribosomal protein S12, and that the pattern of amino acid substitutions in ribosomal protein S12 shows distinct differences in the closely related genera, Wolbachia and Rickettsia.     

Synthesis of protoporphyrin IX induced by 5-aminolevulinic acid in yeast cells in the presence of 2,2;-dipyridyl

5-Aminolevulinic acid (ALA), a precursor of porphyrin synthesis, increased the production of various porphyrin compounds in Candida guilliermondii cells. Metalloporphyrins and protoporphyrin IX (PPIX) were predominantly accumulated, respectively, at ALA concentrations of 0.2-0.4 mM and at those higher than 1.5 mM. 2,2;-Dipyridyl which complexed with bivalent metals significantly increased the content of endogenous PPIX even at ALA concentrations lower than 0.5 mM. Under these conditions, the yeast sensitivity to photodynamic effect of visible light (400-600 nm) dramatically increased due to photosensitization by endogenous PPIX.     

VESICULAR AND BULLOUS DISEASES OF THE SKIN—Cytologic Diagnosis

A new technique, first described by Tzanck, for diagnosis of vesicular and bullous diseases of the skin by cytologic examination of material firmly scraped from lesions has a number of advantages over excision of biopsy specimens. It takes relatively little time, is painless, simple and inexpensive. The method also has value in research and teaching.     

The tight junction as a barrier to cholesterol in canine epithelial cells

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.