Novel surface lipids of diapausing Manduca sexta pupae. Long chain oxoalcohol esters of acetoacetic, hydroxybutyric, and acetic acids

Ester components in the surface wax from diapausing tobacco hornworm pupae, Manduca sexta L., were separated by thin layer chromatography and gas-liquid chromatography, and characterized by infrared spectroscopy and gas-liquid chromatography-mass spectrometry. Three groups of esters were identified as natural derivatives of acetic acid, acetoacetic acid, and 3-hydroxybutyric acid. The major ester fraction was identified as a mixture of C26 (10%), C27 (5%), and C28 (85%) oxoalcohol esters of acetoacetic acid. The major homolog consisted of equal amounts of 11-oxooctacosanyl 3-oxobutanoate and 12-oxooctacosanyl 3-oxobutanoate. Lesser amounts of 11- and 12-oxooctacosanyl and n-octacosanyl esters of acetic and 3-hydroxybutyric acids were also identified. The chain length distributions of these C26, C27, and C28 oxoalcohol and n-primary alcohol ester moieties, as well as the isomeric ratios for the 11- and 12-oxoalcohol isomers, were similar to the oxoaldehydes and unesterified oxoalcohols previously identified by Buckner et al (Buckner, J. S., Nelson, D. R., Haak, H., and Pomonis, J. G. (1984) J. Biol. Chem. 259, 8452-8470) as lipid components of the surface wax of M. sexta pupae.     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Cloning of the y1 Locus of Maize,a Gene Involved in the Biosynthesis of Carotenoids

The y1 gene is one of the genes responsible for the production of [beta]-carotene in the endosperm and leaves of maize. We have cloned a Robertson's Mutator-tagged allele of the y1 gene (y1-mum) by using a Mu3 element as a hybridization probe. We substantiate that the cloned sequence is a portion of the y1 gene by molecular analyses of a revertant of a putative Mutator-induced y1 allele and the incidence of insertions within the cloned y1 sequence from several independently derived Mutator-induced y1 mutant stocks. The y1-mum sequence was used to isolate the standard Y1 allele, which conditions the presence of [beta]-carotene in the endosperm of the maize kernel.     

A SURVEY OF SIZES AND WEIGHTS OF BEMISIA TABACI (HOMOPTERA:ALEYRODIDAE)B BIOTYPE LIFE STAGES FROM FIELD GROWN COTTON AND CANTALOUPES

Abstract Size and weight measurements were made for all the life stages of Bemisia tabaci (Gennadius) B biotype from field grown cotton ( Gossypium hirsutum L.) and cantaloupe ( Cucumis melo L., var. cantalupensis ) in Phoenix, AZ and Fargo, ND, USA in 2000 and 2001. Nymphal volumes were derived from the measurements. The average nymphal volume increase for settled 1 st to the late 4th instar was exponential. The greatest increase in body volume occurred during development from the 3rd to early 4th instar. Nymphs on cotton leaves were wider, but not longer compared with those on cantaloupe. Ventral and dorsal depth ratios of nymphal bodies from 1 st to late 4th instars from cantaloupe leaves were significantly greater compared with those from cotton leaves. During nymphal development from 1st to 4th instar, the average (from the two host species) ventral body half volume increased by nearly 51 times compared with an increase of 28 times for the dorsal body half volume. Adult female and male average lengths, from heads to wing tips, were 1 126 μm and 953 μm, respectively. Average adult female and male weights were 39 and 17 μg, respectively. Average widths, lengths, and weights of eggs from cotton and cantaloupe were, 99 μm, 197 μm, and 0.8 μg, respectively. Average widths, lengths, and weights for exuviae of non-parasitized nymphs from both cotton and cantaloupe were 492 μm, 673 μm, and 1.20 μg, respectively; and widths, lengths, and weights of parasitized nymph exuviae were 452 μm, 665 μm, and 3.62 μg, respectively. Both exuviae from non-parasitized and parasitized nymphs from cotton leaves were wider, longer, and heavier than those from cantaloupe leaves.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.