Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts)

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 ∼ C6:0 ∼ C8:0 < no ceramide < C12:0 < C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 ∼ C6:0 < C2:0 < C12:0 ∼ no ceramide < C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.     

Elucidation of common pharmacophores from analysis of targeted metabolites transported by the multispecific drug transporter-Organic anion transporter1 (Oat1)

Organic anion transporter 1 (Oat1), first identified as NKT, is a multispecific transporter responsible for the handling of drugs and toxins in the kidney and choroid plexus, but its normal physiological role appears to be in small molecule metabolite regulation. Metabolites transported by Oat1 and which are altered in the blood and urine of the murine Oat1 knockout, may serve as templates for further drug design. This may lead to better tissue targeting of drugs or design of Oat1 inhibitors that prolong the half-life of current drugs. Due to the multispecificity of the transporter, 19 of known targeted metabolites have different chemical structures and properties that make constructing a common pharmacophore model difficult. Here we propose an approach that clustered the metabolites into four distinct groups which allowed for the construction of a consensus pharmacophore for each cluster. The screening of commercial molecular databases determined the top candidates whose interaction with Oat1 was confirmed in an experimental model of organic anion transport. Thus, these candidate selections represent potential molecules for further drug design.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.     

Influence of malaria parasite (P. vivax) on erythrocyte aggregation: a study based on dynamic imaging and analysis

Malaria due to P. vivax (PV) is prevalent in many countries. The present work is aimed to determine the cell-cell interaction through formation of aggregates under dynamic conditions. Blood samples are obtained from patients (n=11) suffering from PV malaria, and the normal subjects (n=10) in test tubes containing citrate phosphate dextrose (10:1.4), as an anticoagulant. The signature analysis of infected erythrocytes shows significant alterations in their shape and membrane. For aggregation analysis, erythrocyte suspension in plasma at hematocrit 5%, was placed in a glass chamber and mounted vertically on the stage of the video-microscope system. The aggregate images thus acquired show erythrocytes adhering with each other to form mash-like structures. With increase in parasitaemia, the erythrocytes show hyper-aggregation compared to that of normal cells. By processing of the sequence of recorded images during sedimentation, the various aggregation parameters are obtained. These parameters show that the formed aggregates are compact which produce distinct changes in sedimentation pattern with significantly higher sedimentation velocity compared to that in healthy blood samples. These changes in malaria could partly be responsible for alteration in blood flow through microcirculatory system.     

Effect of different temperature on starch synthase activity in excised grains of wheat cultivars

Excised grains of wheat (Triticum aestivum) varieties HD 2285 (relatively tolerant) and HD 2329 (susceptible type) were incubated for 1 hr at 15 degrees, 25 degrees, 35 degrees and 45 degrees C. In an another treatment, excised grains were incubated for 1 hr at increasing temperature (15 degrees, 25 degrees, 35 degrees and 45 degrees C) continuously, thus exposing the grains to gradual rise in temperature. The above treated grains were then analysed for the activity of soluble starch synthase (SSS) and granule bound starch synthase (GBSS) assayed at 20 degrees C. SSS activity decreased as the pre-exposure temperature was higher, though the tolerant variety showed lesser decrease. Decrease in SSS activity was lesser when excised grains were exposed to gradual rise in temperature from 15 degrees to 45 degrees C as compared to direct exposure to 45 degrees C. Pre-exposure of excised grains to different temperatures however, had no significant effect on GBSS activity.     

Boll weevil (Coleoptera: Curculionidae) survival through cotton gin trash fans

There is concern that cotton gins may serve as loci for reintroduction of boll weevils, Anthonomus grandis grandis Boheman, to eradicated or suppressed zones when processing weevil-infested cotton from neighboring zones. Previous work has shown that virtually all weevils entering the gin in the seed cotton will be removed before they reach the gin stand. Those not killed by the seed cotton cleaning machinery will be shunted alive into the trash fraction, which passes through a centrifugal trash fan before exiting the gin. The objective of this study was to determine survival potential of boll weevils passed through a trash fan. Marked adult weevils were distributed in gin trash and fed through a 82.6-cm (32.5-in.) diameter centrifugal fan operated across a range of fan-tip speeds. A small number of boll weevils were recovered alive immediately after passage through the fan, but all were severely injured and did not survive 24 h. In another experiment, green bolls infested with both adult- and larval-stage weevils were fed through the fan. Several teneral adults survived 24 h, and there was no evidence that fan-tip speed affected either initial survival of weevils, or the number of unbroken boll locks that could harbor an infesting weevil. Thus, designating a minimum fan-tip speed for ensuring complete kill is not possible for the boll weevil. Experiments suggest that a device installed in a gin that partially crushes or cracks bolls open before entering a trash fan will increase mortality, possibly enough that further precautions would be unnecessary.     

Nucleic acid amplification of Mycobacterium tuberculosis complex DNA from archival fine needle aspiration smear scrapings vs. fresh fine needle aspirates of tuberculous lymphadenitis

OBJECTIVE: To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases. STUDY DESIGN: Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears. RESULTS: Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases. CONCLUSION: Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.     

Development and population growth of Lygus hesperus on selected weed hosts, artificial diet and cotton in the laboratory

Laboratory studies were conducted to obtain basic biological information of Lygus hesperus Knight (Hemiptera: Miridae) on alfalfa (Medicago sativa L.), pigweed (Amaranthus palmeri S. Wats.), Russian thistle (Salsola iberica Sennen and Pau), green bean (Phaseolus vulgaris L.), artificial diet, and cotton (Gossypium hirsutum L.) square and cotton boll at 27 degrees C. The nymphal developmental duration was 28.8 d on cotton bolls, nearly twice longer, compared with approximately 15 d in other hosts. The nymphal survivorship was significantly lowest on the cotton boll (18%), whereas the survivorship in other hosts ranged from 33% (cotton square) to 93% (green bean). The average total nymphal survivorship was approximately 60% and the most nymphal mortalities occurred on the first three instars. The life-table parameters were obtained only in cotton square, artificial diet, green bean, and alfalfa. The average adult longevity in artificial diet was the shortest (18.7 d),with the longest longevity observed (36.1 d) on green beans. The intrinsic rates of population increase (r(m)) were much higher in artificial diet (0.0771) and green beans (0.0671) than in alfalfa (0.0327) and cotton square (0.0317). Although statistically significant, the difference in r(m) values between alfalfa and cotton square was very small, indicating the similarity in reproductive suitability of cotton square and alfalfain a no-choice test. Thus, even though cotton is not a preferred host, when blooming alfalfa and roadside weeds are mowed in the Texas High Plains during June-July, it is the most likely time that Lygus bugs may move from alfalfa and other host plants into adjacent cotton fields and become effective pest of actively fruiting cotton.     

Serum-free hybridoma culture: ethical, scientific and safety considerations

Despite considerable progress in the development of cell culture techniques, including the development of the serum- and protein-free media that now routinely support hybridoma and mammalian cell growth, fetal bovine serum (FBS) supplemented media are still commonly used: a practice that raises ethical, scientific and safety concerns. The use of FBS in hybridoma culture media is examined here, with regards to the development and production of monoclonal antibodies (mAbs), and it is our recommendation that researchers adopt serum-free cell culture methods to reduce animal use in this area.     

Photosynthetic response of wheat and sunflower cultivars to long-term exposure of elevated carbon dioxide concentration

Wheat (Triticum aestivum L. cv. HD 2329 and DL 1266-5) and sunflower (Helianthus annuus L. cv. MSFH 17 and MRSF 1754) plants were grown in field under atmospheric (360±10 cm³ m⁻³, AC) and elevated (650±50 cm³ m⁻³, EC) CO₂ concentrations in open top chambers for entire period of growth and development till maturity. Net photosynthetic rate (P _N) of wheat cvs. when compared at the same internal CO₂ concentration (C _i), by generating P _N/C _i curves, showed lower P _N in EC plants than in AC ones. EC-grown wheat cultivars also showed a lesser response to irradiance than AC plants. In sunflower cultivars, P _N/C _i curves and irradiance response curves were not significantly different in AC and EC plants. CO₂ and irradiance responses of photosynthesis, therefore, further revealed a down-regulation of _{P N} in wheat but not so in sunflower under long-term CO₂ enrichment. Wheat cvs. accumulated in leaves mostly sugars, whereas sunflower accumulated mainly starch. This further strengthened the view that accumulation of excess assimilates in the leaves under EC as starch is not inhibitory to _{P N}.