Cyclodextrin Treatment Research Articles

Effect of cholesterol-loaded cyclodextrin treatment on boar sperm cryopreservation.

This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality. In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing. The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05). The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar.

Quorum quenching effect of cyclodextrins on the pyocyanin and pyoverdine production of Pseudomonas aeruginosa

Various virulence determinants in Pseudomonas aeruginosa are regulated by the quorum sensing (QS) network producing and releasing signalling molecules. Two of these virulence determinants are the pyocyanin and pyoverdine, which interfere with multiple cellular functions during infection. The application of QS-inhibiting agents, such as cyclodextrins (CDs), appears to be a promising approach. Further to method development, this research tested in large-volume test systems the effect of α- and β-CD (ACD, BCD) at 1, 5, and 10 mM concentrations on the production of pyocyanin in the P. aeruginosa model system. The concentration and time-dependent quorum quenching effect of native CDs and their derivatives on pyoverdine production was tested in a small-volume high-throughput system. In the large-volume system, both ACD and BCD significantly inhibited pyocyanin production, but ACD to a greater extent. 10 mM ACD resulted in 58% inhibition, while BCD only ~40%. Similarly, ACD was more effective in the inhibition of pyoverdine production; nevertheless, the results of RMANOVA demonstrated the significant efficiency of both ACD and BCD, as well as their derivatives. Both the contact time and the cyclodextrin treatments significantly influenced pyoverdine production. In this case, the inhibitory effect of ACD after 48 h at 12.5 mM was 57%, while the inhibitory effect of BCD and its derivatives was lower than 40%. The high-level significant inhibition of both pyocyanin and pyoverdine production by ACD was detectable. Consequently, the potential value of CDs as QS inhibitors and the antivirulence strategy should be considered.Keypoints• Applicability of a simplified method for quantification of pyocyanin production was demonstrated.• The cyclodextrins significantly affected the pyocyanin and pyoverdine production.• The native ACD exhibited the highest attenuation in pyoverdine production.

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Differences in proteome perturbations caused by the Wolbachia strain wAu suggest multiple mechanisms of Wolbachia-mediated antiviral activity

Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue virus (DENV) and other RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for DENV control. The degree of virus inhibition varies between Wolbachia strains. Density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar intracellular densities but only wAu produces strong DENV inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel/wMelPop in Ae. aegypti. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines carrying strains wAu and wAlbA: with the hypothesis that differences in perturbations may underline Wolbachia-mediated antiviral activity. Surprisingly, wAu-carrying midguts not only showed distinct proteome perturbations when compared to non-Wolbachia carrying and wAlbA-carrying midguts but also wMel-carrying midguts. There are changes in RNA processing pathways and upregulation of a specific set of RNA-binding proteins in the wAu-carrying line, including genes with known antiviral activity. Lipid transport and metabolism proteome changes also differ between strains, and we show that strain wAu does not produce the same cholesterol sequestration phenotype as wMel. Moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. Together these results suggest that wAu could show unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p<.05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p<.05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p<.05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Cellular Effects of Cyclodextrins: Studies on HeLa Cells.

Cyclodextrins are high molecular weight, hydrophilic, cyclic, non-reducing oligosaccharides, applied as excipients for the improvement of the solubility and permeability of insoluble active pharmaceutical ingredients. On the other hand, beta-cyclodextrins are used as cholesterol sequestering agents in life sciences. Recently, we demonstrated the cellular internalization and intracellular effects of cyclodextrins on Caco-2 cells. In this study, we aimed to further investigate the endocytosis of (2-hydroxylpropyl)-beta-(HPBCD) and random methylated-beta-cyclodextrin (RAMEB) to test their cytotoxicity, NF-kappa B pathway induction, autophagy, and lysosome formation on HeLa cells. These derivatives were able to enter the cells; however, major differences were revealed in the inhibition of their endocytosis compared to Caco-2 cells. NF-kappa B p65 translocation was not detected in the cell nuclei after HPBCD or RAMEB pre-treatment and cyclodextrin treatment did not enhance the formation of autophagosomes. These cyclodextrin derivates were partially localized in lysosomes after internalization.

Quality stability of clear pomegranate juice treated with cyclodextrin.

Protective effect of cyclodextrin (CD) on some quality characteristics of clear pomegranate juice (CPJ) was investigated. CPJ with no CD addition (Control) and CPJ samples, subjected to different type (β-CD and Hydroxy propyl (HP-β-CD) and concentration (0.5, 1% and 2%) combinations of CD, were stored at 25°C for 3months. The changes in color characteristics (polymeric color and total color difference - ΔE), total phenolic content (TPC) and antioxidant activity (AA) of all PJ samples monitored at 1month intervals. Although overall color change of the PJ samples was hardly perceived visually, none of the treatments were successful to protect the initial color of clear PJ technically (P > 0.05). Among the CD treatments, HP-β-CD with a concentration of 0.5% was found effective to prevent TPC and AA losses of PJ (P < 0.05). On the other hand, β-CD addition did not cause any improvement in protecting color, TPC and AA of clear PJ during storage, compared to control juice.

Impact of cyclodextrin treatment on composition and sensory properties of lingonberry (Vaccinium vitis-idaea) juice

Lingonberry (Vaccinium vitis-idaea L.) juice was treated with 10 g/L cyclodextrins (CD) or gelatin as potential means to improve the stability of bioactive compounds and to modify the sensory properties. Before and after the treatment acids, sugars, and phenolic compounds (anthocyanins, flavonols, phenolic acids, proanthocyanidins) of the juices were analyzed with chromatographic and mass spectrometric methods. Forty-six phenolic compounds were identified or tentatively identified, of which twenty-four were A-type or B-type proanthocyanidins with degree of polymerization of 1–9. Juices treated with β-CD had the highest contents of phenolic compounds (phenolic acids 17.9 mg/100 mL, flavonols 47.5 mg/100 mL, anthocyanins 21.0 mg/100 mL, and proanthocyanidins 114.2 mg/mL), whereas gelatin treatment resulted in significantly lowered content of the compounds (phenolic avids 16.3 mg/100 mL, flavonols 43.50 mg/100 mL, anthocyanins 19.7 mg/100 mL, and proanthocyanidins 81.7 mg/mL). Taste and astringency attributes of the juices were investigated with untrained panelists (n = 40). The lingonberry juices were perceived as notably sour, bitter and astringent. CD or gelatin treatments alone had less effect on the astringency of the juice in comparison to their use in combination. The results suggest that, CDs may be utilized as protectants of the phenolic compounds, especially anthocyanins, in lingonberry juices.

Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line

In recombinant protein production, over-expressed genes induce unfolded protein response (UPR), overloaded protein aggregation in endoplasmic reticulum and its expansion. In this study, we have used 16 chemicals to improve erythropoietin production in engineered CHO cells and tried to study the mechanism of reducing protein aggregation in each treatment. Endoplasmic reticulum expansion was studied through endoplasmic reticulum specific labeling with utilizing fluorescent glibenclamide and its molecular chaperones expression were studied by real-time polymerase chain reaction. The increase in the mRNA level of EPO and endoplasmic reticulum chaperones GRP78/BiP, XBP1, ATF6, and ATF4 in different chemical treatments were not related to ER expansion. On the other hand, ER expansion in beta alanine, beta cyclodextrin and taurine treatments resulted in increased EPO secretion. Dramatically increase in EPO expression in conjugated linoleic acid, spermidine, trehalose, and maltose (19, 20, 16, and 19-fold, respectively) did not increase erythropoietin productivity, but betaine which did not caused ER expansion, with minor increase in EPO gene expression increase EPO productivity. The results indicated that betaine increase EPO secretion in engineered CHO cell line without relation to ER expansion and molecular chaperones expression.

Abstract 220: Targeting Cholesterol Efflux, Autophagy, and Inflammation to Prevent Atherosclerosis

Introduction: The NLRP3 inflammasome and Toll-like receptor signaling is activated in advanced human atherosclerotic plaques, while autophagy and reverse cholesterol transport (RCT) become dysfunctional. Simultaneous targeting of these pathways can prevent CVD. Objectives: To determine if Miltefosine can prevent atherosclerosis by inducing RCT/autophagy and dampening inflammation. Methods and results: Here, we report that Miltefosine, an anti-leishmanial drug, acts to decrease atherosclerosis in vivo, and that it induced RCT/autophagy while dampening TLR signaling and NLRP3 inflammasome activity. Miltefosine treatment of macrophages disrupted lipid rafts; ~26 % decrease vs. control via alexa647-CTB binding by flow-cytometry, (p&lt;0.005, n=4), and increased ABCA1 mediated cholesterol efflux to apoA1; ~20% increase in treated vs. control (p&lt;0.001, n=3). Macrophages treated with Miltefosine vs. control exhibited a marked increase in autophagosomes, indicated by p62 and LC3 staining puncta. Autophagic degradative flux was not inhibited by Miltefosine. Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester:free cholesterol) ratio (p&lt;0.005, n=3). Miltefosine treated vs. control macrophages showed ~75% reduction in pro-IL1β mRNA levels upon LPS induction (p&lt;0.05, n=3). Miltefosine potently inhibited NLRP3 inflammasome assembly upon LPS/ATP treatment leading to ~70% reduction in ASC1 speck positive cells. Gasdermin D mediated release of mature IL1β was reduced by ~80% in Miltefosine treated vs. controls (p&lt;0.01, n=3), while only ~20% reduction was observed by cyclodextrin treatment, indicating that inflammasome inhibition by Miltefosine was not only due to cholesterol depletion. In vivo pilot studies showed that Miltefosine added to a chow diet increased RCT to plasma (~70% increase in 24h vs. control), and feces (~60% increase in 24h vs. control, p&lt; 0.005, n=5) in C57BL6 mice. Atherosclerotic lesions were =50% smaller in apoE -/- mice fed chow diet + Miltefosine vs. controls (p&lt;0.02, n=5). Conclusion: Miltefosine induced RCT and autophagy while dampening TLR signaling and NLRP3 inflammasome activity, and it decreased atherosclerosis lesion burden in mice.

Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment.

Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIα, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.

Study on properties of cotton fabric incorporated with diammonium phosphate flame retardant through cyclodextrin and 1,2,3,4-butane tetracarboxylic acid binding system

The chemical and physical structure of cyclodextrin makes it attractive for use in flame retardant finishing for improving the functional properties and durability of the finished fabric. The suitability of cyclodextrins for grafting using viable techniques that could be reproduced was studied. The flame retardant diammonium phosphate was applied on cyclodextrin-grafted cotton fabric and grafted cum bonded with 1,2,3,4-butane tetracarboxylic acid fabric. The modified monochlorotriazinyl-β-cyclodextrin gives more percentage of grafting (2.7%) under normal processing conditions used for cotton fabric. The effect of treatments like cyclodextrin grafting and 1,2,3,4-butane tetracarboxylic acid treatment on grafted fabric were analysed for diammonium phosphate flame retardant finishing. The whiteness and loss of strength caused by the cyclodextrin grafting was within acceptable limits and the hand value alteration was very less. The flammability behaviour and durability after 1, 5, 10, 20 and 40 washing cycles were determined. The results reveal that the flame retardant property of cyclodextrin treatment enhances the durability of the fabric from non-durable to semi-durable, whereas the diammonium phosphate treatment on 1,2,3,4-butane tetracarboxylic acid/cyclodextrin-grafted fabric changes the flame retardant from non-durable to durable. The evolution of gases during the burning of untreated and treated samples was analysed and the results show that the cyclodextrin-grafted fabric masks the evolving gases to some extent during burning and also that there was less release of formaldehyde in grafted samples compared with ungrafted diammonium phosphate finished sample.

Development of targeted therapies for Parkinson's disease and related synucleinopathies

Therapeutic efforts in neurodegenerative diseases have been very challenging, particularly due to a lack of validated and mechanism-based therapeutic targets and biomarkers. The basic idea underlying the novel therapeutic approaches reviewed here is that by exploring the molecular basis of neurodegeneration in a rare lysosomal disease such as Gaucher's disease (GD), new molecular targets will be identified for therapeutic development in common synucleinopathies. Accumulation of α-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, suggesting that improved clearance of α-synuclein may be of therapeutic benefit. To achieve this goal, it is important to identify specific mechanisms and targets involved in the clearance of α-synuclein. Recent discovery of clinical, genetic, and pathological linkage between GD and PD offers a unique opportunity to examine lysosomal glucocerebrosidase, an enzyme mutated in GD, for development of targeted therapies in synucleinopathies. While modulation of glucocerebrosidase and glycolipid metabolism offers a viable approach to treating disorders associated with synuclein accumulation, the compounds described to date either lack the ability to penetrate the CNS or have off-target effects that may counteract or limit their capabilities to mediate the desired pharmacological action. However, recent emergence of selective inhibitors of glycosphingolipid biosynthesis and noninhibitory pharmacological chaperones of glycosphingolipid processing enzymes that gain access to the CNS provide a novel approach that may overcome some of the limitations of compounds reported to date. These new strategies may allow for development of targeted treatments for synucleinopathies that affect both children and adults.

2-Hydroxypropyl-β-cyclodextrin Promotes Transcription Factor EB-mediated Activation of Autophagy

2-Hydroxypropyl-β-cyclodextrin (HPβCD) is a Food and Drug Administration-approved excipient used to improve the stability and bioavailability of drugs. Despite its wide use as a drug delivery vehicle and the recent approval of a clinical trial to evaluate its potential for the treatment of a cholesterol storage disorder, the cellular pathways involved in the adaptive response that is activated upon exposure to HPβCD are still poorly defined. Here, we show that cell treatment with HPβCD results in the activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and in enhancement of the cellular autophagic clearance capacity. HPβCD administration promotes transcription factor EB-mediated clearance of proteolipid aggregates that accumulate due to inefficient activity of the lysosome-autophagy system in cells derived from a patient with a lysosomal storage disorder. Interestingly, HPβCD-mediated activation of autophagy was found not to be associated with activation of apoptotic pathways. This study provides a mechanistic understanding of the cellular response to HPβCD treatment, which will inform the development of safe HPβCD-based therapeutic modalities and may enable engineering HPβCD as a platform technology to reduce the accumulation of lysosomal storage material.

Caveolin-1 in Lipid Rafts Interacts with Dengue Virus NS3 during Polyprotein Processing and Replication in HMEC-1 Cells

Lipid rafts are ordered microdomains within cellular membranes that are rich in cholesterol and sphingolipids. Caveolin (Cav-1) and flotillin (Flt-1) are markers of lipid rafts, which serve as an organizing center for biological phenomena and cellular signaling. Lipid rafts involvement in dengue virus (DENV) processing, replication, and assembly remains poorly characterized. Here, we investigated the role of lipid rafts after DENV endocytosis in human microvascular endothelial cells (HMEC-1). The non-structural viral proteins NS3 and NS2B, but not NS5, were associated with detergent-resistant membranes. In sucrose gradients, both NS3 and NS2B proteins appeared in Cav-1 and Flt-1 rich fractions. Additionally, double immunofluorescence staining of DENV-infected HMEC-1 cells showed that NS3 and NS2B, but not NS5, colocalized with Cav-1 and Flt-1. Furthermore, in HMEC-1cells transfected with NS3 protease, shown a strong overlap between NS3 and Cav-1, similar to that in DENV-infected cells. In contrast, double-stranded viral RNA (dsRNA) overlapped weakly with Cav-1 and Flt-1. Given these results, we investigated whether Cav-1 directly interacted with NS3. Cav-1 and NS3 co-immunoprecipitated, indicating that they resided within the same complex. Furthermore, when cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release. These data demonstrate the essential role of caveolar cholesterol-rich lipid raft microdomains in DENV polyprotein processing and replication during the late stages of the DENV life cycle.

Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling

Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1(-/-) mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-β-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate

Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and β-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom.

Membrane phase behavior during cooling of stallion sperm and its correlation with freezability

Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures, from 30-10°C, whereas freezing-induced dehydration of the cells causes a more severe phase transition to a highly ordered gel phase. Sperm from individual stallions showed significant differences in post-thaw progressive motility, percentages of sperm with abnormal cell morphology, and chromatin stability. The biophysical membrane properties evaluated in this study, however, did not show clear differences amongst stallions with differences in sperm freezability. Cyclodextrin treatment to remove cholesterol from the cellular membranes increased the cooperativity of the suprazero phase transition, but had little effects on the subzero membrane phase behavior. In contrast, freezing of sperm in the presence of protective agents decreased the rate of membrane dehydration and increased the total extent of dehydration. Cryoprotective agents such as glycerol decrease the amount of energy needed to transport water across cellular membranes during freezing.

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ~40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2(-/-) and npc1(-/-) mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.

Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus

Cellular membrane cholesterol has been shown to support various membrane proteins. However, the role and function of membrane cholesterol in viral production are still unclear. Here, we investigated the effects of cholesterol depletion from the cell membrane on the production of hemagglutinating virus of Japan (HVJ; Sendai virus). Cholesterol depletion from LLC-MK2 cells by methyl-beta cyclodextrin treatment resulted in a marked increase in the production of both HVJ from the infected cells and virus-like particles from M-gene-transfected cells. The HVJ produced from cholesterol-depleted cells possessed a reduced amount of envelope cholesterol and showed a rather wide range of particle sizes and amount of envelope protein compared to HVJ produced from untreated cells. Direct depletion of envelope cholesterol from HVJ significantly impaired its infectivity, even without a change in envelope protein composition. These results suggest that membrane cholesterol plays important roles in regulating the production of infectious HVJ.