Heat Stability Studies Research Articles

Thermostability and Immunostimulatory Activity of Cationic Liposome-based Newcastle Disease Virus Vaccine

Cold storage is a huge challenge in maintaining vaccine potency especially in low and middle income countries (LMIC). The aim of this research was to investigate the heat and freeze-thaw stability of Newcastle disease virus (NDV) vaccine encapsulated in 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) cationic liposomes as well as its immunostimulatory activity. DOTAP cationic liposomes were prepared using the thin-film hydration technique and characterized for morphology, particle size, polydispersity index, zeta potential and encapsulation efficiency. Experimental birds were challenged with Herts 33 velogenic strain of NDV. Haemagglutination inhibition test was used to assess the formation of antibodies in the birds. The cationic liposomes were further stored at 28 oC or subjected to freeze-thaw cycles for 7 weeks and then assessed for stability using the same parameters of particle size, polydispersity index, zeta potential, morphology, antibody titre or differential white blood cell count. Using TEM, the morphology of the liposomes was found to be spherical, and using Malvern Zetasizer, particle size was in the nanosize range. Zetapotential and polydispersity indices were positive and below 0.1. The HI test showed that the DOTAP cationic liposomes stimulated a higher titre of antibodies than the commercial vaccine. Heat and freeze-thaw stability studies after 7 weeks of storage showed slight aggregation and elevated particle sizes and wider distribution. The HI titer was appreciably lower after secondary immunization with the DOTAP cationic liposomes stored at 28 oC but elevated for the product kept at freeze-thaw conditions. The immunized birds were 100 % protected from the virulent strain of NDV. In conclusion, the development of the DOTAP-cationic liposomes encapsulating NDV vaccine administered orally was found to be more stable under freeze-thaw conditions than when it was exposed to heat for 7 weeks. The liposome-based vaccine had more immunostimulatory effects than the commercial sample.

Light and hydrogen peroxide dual-responsive DNA interstrand crosslink precursors with potent cytotoxicity

Arylboronic acid/esters and phenyl selenides-based quinone methide (QM) precursors were reported to induce DNA interstrand crosslink (ICL) formation upon reaction with the inherently high concentrations of H2O2 in cancer cells. However, some normal cells (such as macrophages) also contain high-levels of H2O2, which may interfere with precursors’ selectivity. In order to enhance the spatiotemporal specificity by the photolysis, we developed photo- and H2O2– dual-responsive DNA ICL precursors 1–3, bearing a photo-responsive coumarin moiety and a H2O2 inducible phenyl selenide group. Precursors 1–3 are efficiently activated by photoirradiation and H2O2 to generate reactive QMs crosslinking DNA. Moreover, the reactivity of precursors can be modulated by the introduction of aromatic substituents (OMe, F), and the electron donating group (OMe) displays a more pronounced promoting effect on DNA ICL formation. A subsequent piperidine heat stability study confirmed that the formed QMs primarily alkylate dAs, dGs and dCs in DNA. Furthermore, 1–3 inhibit lung cancer cell (H1299) growth by inducing DNA damage and producing toxic reactive oxygen species (ROS) upon photolysis of released coumarin. This study illustrates the potent cytotoxicity achieved by novel photo/H2O2 dual-responsive QM precursors 1–3, affording a novel strategy for the development of inducible DNA interstrand cross-linkers.

Photoinduced DNA Interstrand Cross-Linking by 1,1'-Biphenyl Analogues: Substituents and Leaving Groups Combine to Determine the Efficiency of Cross-Linker.

Two series of 1,1'-biphenyl analogues with various leaving groups (L=OAc, OCH3 , OCHCH=CH2 , OCH2 Ph, SPh, SePh, and Ph3 P+ ) were synthesized. Their reactivity towards DNA and the reaction mechanism were investigated by determining DNA interstrand cross-link (ICL) efficiency, radical and carbocation formation, and the cross-linking reaction sites. All compounds induced DNA ICL formation upon 350 nm irradiation via a carbocation that was generated from oxidation of the corresponding free radicals. The ICL efficiency and the reaction rate strongly depended on the combined effect of the leaving group and the substituent. Among all compounds tested, the high ICL efficiency (30-43 %) and fast reaction rate were observed with compounds carrying a nitrophenyl group and acetate (2 a), ether (2 b and 2 c), or triphenylphosphonium salt (2 g) as leaving groups. Most compounds with a 4-methoxybenzene group showed similar DNA ICL efficiency (≈30 %) with a slow DNA cross-linking reaction rate. Both cation trapping and free radical trapping adducts were detected in the photo activation process of these compounds, which provided direct evidence for the proposed mechanism. Heat stability study in combination with sequence study suggested that these photo-generated benzyl cations alkylate DNA at dG, dA, and dC sites.

Photoinduced DNA Interstrand Cross-Linking by Benzene Derivatives: Leaving Groups Determine the Efficiency of the Cross-Linker

We have synthesized and characterized two small libraries of 2-OMe or 2-NO2-benzene analogues 2a-i and 3a-i containing a wide variety of leaving groups. Irradiation of these compounds at 350 nm generated benzyl radicals that were spontaneously oxidized to benzyl cations directly producing DNA interstrand cross-links (ICLs). Compounds with a 2-methoxy substituent showed a faster cross-linking reaction rate and higher ICL efficiency than the corresponding 2-nitro analogues. Apart from the aromatic substituent, the benzylic leaving groups greatly affected DNA cross-linking efficiency. Higher ICL yields were observed for compounds with OCH3 (3b), OCH2Ph (3d), or Ph3P+ (3i) as leaving groups than those containing OAc (3a), NMe2 (3e), morpholine (3f), OCH2CH═CH2 (3c), SPh (3g), or SePh (3h). The heat stability study of the isolated ICL products indicated that dGs were the preferred alkylation sites in DNA for the benzyl cations produced from 2a-i, 3c, and 3e-i while 3a (L = OAc), 3b (L = OMe), and 3d (L = OCH2Ph) showed a similar photoreactivity toward dGs and dAs. Although the photogenerated benzyl cations alkylated dG, dC, and dA, ICL assay with variation of DNA sequences showed that the ICL reaction occurred with opposing dG/dC but not with staggered dA/dA.

A potent novel vaccine adjuvant based on straight polyacrylate

A structure-activity study was conducted to identify the structural characteristics underlying the adjuvant activity of straight (i.e. non-crosslinked) polyacrylate polymers (PAAs) in order to select a new PAA adjuvant candidate for future clinical development. The study revealed that the adjuvant effect of PAA was mainly influenced by polymer size (Mw) and dose. Maximal effects were obtained with large PAAs above 350 kDa and doses above 100 μg in mice. Small PAAs below 10 kDa had virtually no adjuvant effect. HPSEC analysis revealed that PAA polydispersity index and ramification had less impact on adjuvanticity. Heat stability studies indicated that residual persulfate could be detrimental to PAA stability. Hence, this impurity was systematically eliminated by diafiltration along with small Mw PAAs and residual acrylic acid that could potentially affect product safety, potency and stability. The selected PAA, termed SPA09, displayed an adjuvant effect that was superior to that of a standard emulsion adjuvant when tested with CMV-gB in mice, even in the absence of binding to the antigen. The induced immune response was dominated by strong IFNγ, IgG2c and virus neutralizing titers. The activity of SPA09 was then confirmed on human cells via the innate immune module of the human MIMIC® system.

Effects of Voxelotor (GBT440) Therapy on Laboratory Testing of Sickle Cell Patients

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

Uniaxial Expansion of the 2D Ruddlesden-Popper Perovskite Family for Improved Environmental Stability.

The unique hybrid nature of 2D Ruddlesden-Popper (R-P) perovskites has bestowed upon them not only tunability of their electronic properties but also high-performance electronic devices with improved environmental stability as compared to their 3D analogs. However, there is limited information about their inherent heat, light, and air stability and how different parameters such as the inorganic layer number and length of organic spacer molecule affect stability. To gain deeper understanding on the matter we have expanded the family of 2D R-P perovskites, by utilizing pentylamine (PA)2(MA) n-1Pb nI3 n+1 ( n = 1-5, PA = CH3(CH2)4NH3+, C5) and hexylamine (HA)2(MA) n-1Pb nI3 n+1 ( n = 1-4, HA = CH3(CH2)5NH3+, C6) as the organic spacer molecules between the inorganic slabs, creating two new series of layered materials, for up to n = 5 and 4 layers, respectively. The resulting compounds were extensively characterized through a combination of physical and spectroscopic methods, including single crystal X-ray analysis. High resolution powder X-ray diffraction studies using synchrotron radiation shed light for the first time to the phase transitions of the higher layer 2D R-P perovskites. The increase in the length of the organic spacer molecules did not affect their optical properties; however, it has a pronounced effect on the air, heat, and light stability of the fabricated thin films. An extensive study of heat, light, and air stability with and without encapsulation revealed that specific compounds can be air stable (relative humidity (RH) = 20-80% ± 5%) for more than 450 days, while heat and light stability in air can be exponentially increased by encapsulating the corresponding films. Evaluation of the out-of-plane mechanical properties of the corresponding materials showed that their soft and flexible nature can be compared to current commercially available polymer substrates (e.g., PMMA), rendering them suitable for fabricating flexible and wearable electronic devices.

Study of spontaneous combustion mechanism and heat stability of sulfide minerals powder based on thermal analysis

The spontaneous combustion mechanism of sulfide minerals powder is studied by simultaneous thermal analysis. The chemical reactions of pyrites in the atmospheres of air and nitrogen are deduced and the corresponding reaction products are characterized by the Raman spectroscopy. The characteristic temperatures of the pyrite reaction process are discussed in different conditions. The results show that the process of oxidation spontaneous combustion can be divided into three stages. Fe3FeSiO4(OH)5 in the minerals is decomposed at low temperature in the oxidation process. FeS2, the main component of the mineral, is mainly oxidized in the stage of oxidation spontaneous combustion. However, FeS2 will be decomposed under heating in nitrogen atmosphere. Thermal stability of pyrite in air is lower than that in nitrogen, and its thermal stability will be improved by increasing heating rate and particle size.

Synergistic versus Additive Effect of Active Molecular Chitosan with Biochemical Compounds of Sorghum bicolor

The synergistic or additive effect of active molecular chitosan (AMC) with Sorghum bicolor ethanol extract (ES) and fermentative supernatant was invesigated in this study. ES had 29% less content of phenolic compounds, 78% less contents of total carbohydrates containing reducing sugars, and 56% less tannin contents than that of S. bicolor fermentative supernatant (SF). It has shown the significantly higher antioxidant activity than SF and other fermantative substances (S and Y). These results suggest that flavonoid in the ES may be the key molecule as it contains 28% higher concentration of flavonoids than SF and Y substances and hence ES showed the remarkablely higher specific activity of whitenning effect. Further more SF was selected for heat stability studies, tyrosinase inhibitory, and antibacterial activity in the presence or absence of active molecular chitosan (AMC). Although less significant synergistic antibacterial activity of SF toward Candida albicans in the presence of AMC was observed, both substances still can be useful as antioxidant or whitenning substances in combination with AMC. whereas a strong antibacterial activity of AMC toward Candica albicans was observed, and also is one of the ingredients in functional food and cosmaceutical compounds with potential health-promoting properties.

Inheritance of heat stable esterase in near isogenic lines and functional classification of esterase in silkworm Bombyx mori

Esterases are ubiquitous in living organisms and perform multiple functions in animals, plants, insects and microorganisms. Insect esterases broadly perform physiological and defense functions. The present work aims towards identifying heat stable esterase in the hemolymph of near isogenic lines (NILs) and their parents, and further to classify esterases based on substrate-inhibition reactions in silkworm, Bombyx mori. Five different α-esterases viz. Est-1, Est-2, Est-3, Est-4 and Est-5 were observed in this study. Of them, Est-1 and Est-2 were monomorphic and, Est-3, Est-4 and Est-5 were polymorphic. Heat stability studies revealed Est-2 and Est-3 as heat stable and, Est-1, Est-4 and Est-5 as heat liable. Through inhibition analysis, Est-1 was identified as cholinesterase and Est-5 as carboxylesterase because their activity was totally inhibited, respectively, by eserine sulphate and phenylmethylsulfonyl fluoride (PMSF) while Est-3 was inhibited by both the inhibitors. The Est-2 and Est-4 were unaffected by both PMSF and eserine sulphate. The isozyme patterns of breed specific as well as heat stable esterases supports the variations in the survival percentage of silkworm breeds at high temperatures. This study enhances the knowledge on esterase-mediated thermotolerance in B. mori.

Nanoblends of novel polyesters with polyaniline: Conductivity and heat-stability studies

The present work initially deals with the fabrication of a new generation of polyesters (bearing thiophene, azomethine, thiourea and ether moieties) and subsequent blending with polyaniline (PAN) exhibiting extremely valuable nanoblend characteristics. Few poly(azomethine-thiourea-ester)s (PATEs), by the polycondensation of a new diol 4,4′-oxydiphenyl bis(( E)-1-(4-hydroxobenzylidene)thiourea) with thiophene-2,5-dicarbonyl/terephtaloyl chloride, were synthesized. The polymers obtained were characterized by Fourier-transform infrared, proton-nuclear magnetic resonance, solubility, solution viscosity, gel permeation chromatography, thermogravimetric analysis, differential scanning calorimetry and wide-angle x-ray diffraction. The polymers exhibited high molar mass (77–89 × 103) and thermal stability ( T10 528–531°C, glass transition temperature ( Tg) 265–268°C). Then the electrically conducting blends, of PAN dispersed in the PATEs matrix, were prepared by mash-blending and melt-blending techniques. Materials obtained by conventional melt-blending approach generated an efficient conductive network compared with those produced by mash blending. Field emission scanning electron microscopy images displayed nanoblend morphology of the melt-blended system, owing to increased physical interactions (hydrogen bonding and π–π stacking) between the two polymers. Moreover, miscible blends of thiophene-based PATE/PAN led to superior conductivity (1.5–2.1 S/cm) and heat stability ( T10 503°C) even at low PAN concentration relative to previous thiophene, azomethine or PAN-based structures.

A Two-Dimensional Numerical Study of Hydrodynamic, Heat and Mass Transfer and Stability in a Salt Gradient Solar Pond

In this work, the problem of hydrodynamic, heat and mass transfer and stability in a salt gradient solar pond has been numerically studied by means of computational fluid dynamics in transient regime. The body of the simulated pond is an enclosure of height H and length L wherein an artificial salinity gradient is created in order to suppress convective motions induced by solar radiation absorption and to stabilize the solar pond during the period of operation. Here we show the distribution of velocity, temperature and salt concentration fields during energy collection and storage in a solar pond filled with water and constituted by three different salinity zones. The bottom of the pond is blackened and the free-surface is subjected to heat losses by convection, evaporation and radiation while the vertical walls are adiabatic and impermeable. The governing equations of continuity, momentum, thermal energy and mass transfer are discretized by finite–volume method in transient regime. Velocity vector fields show the presence of thin convective cells in the upper convective zone (UCZ) and large convective cells in the lower convective zone (LCZ). This study shows the importance of buoyancy ratio in the decrease of temperature in the UCZ and in the preservation of high temperature in the LCZ. It shows also the importance of the thickness of Non-Convective Zone (NCZ) in the reduction of the upwards heat losses.

Biodegradation of phenanthrene by Alcaligenes sp. strain PPH: partial purification and characterization of 1-hydroxy-2-naphthoic acid hydroxylase

Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of ∼34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid-ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained ∼90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The K(m) for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 μM, respectively), suggesting dual specificity for the coenzyme. The K(m) for FAD was determined to be 4.7 μM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1-hydroxylase.

Simple, Selective, and Rapid Quantification of 1‐Deoxynojirimycin in Mulberry Leaf Products by High‐Performance Anion‐Exchange Chromatography with Pulsed Amperometric Detection

1-Deoxynojirimycin (DNJ) occurs in mulberry and other plants and is a highly potent glycosidase inhibitor reported to suppress blood glucose levels, thus preventing diabetes. Derivatization is required for quantification of DNJ upon use of spectral detection methods. Because of this difficulty, the DNJ contents of mulberry-based food products are rarely stated, even if DNJ is their active component. A simple, selective, and rapid method of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to quantify DNJ in mulberry-based food products was developed. Stability testing of DNJ under heat treatment was also performed. A water extract of mulberry tea sample was subjected to HPAEC-PAD in a CarboPac MA1 column with a sodium hydroxide gradient. DNJ was clearly separated at a retention time of 7.26 min without interference and was selectively detected in the water extract. The detection limit was 5 ng. Heat stability studies suggested that DNJ was heat stable. HPAEC-PAD was not subject to interference, was highly selective for DNJ, and was superior to other high-performance liquid chromatography (HPLC) techniques in terms of sample preparation, resolution, and sensitivity. The method allowed simple, selective, and rapid analysis of DNJ in food matrices and might be useful for development of mulberry-based food products. Heat treatment could be an option for sterilizing mulberry-based products.

Simultaneous Determination of Trans-Cinnamaldehyde and Benzaldehyde in Different Real Samples by Differential Pulse Polarography and Study of Heat Stability of Trans-Cinnamaldehyde

A simple, sensitive, and accurate differential pulse polarography method for simultaneous determination of trans-cinnamaldehyde and benzaldehyde in food and drug samples were developed. DC polarography, CV, and coulometric techniques were used for investigation the electrochemical behavior of both compounds. In phosphate buffer (pH = 8.2) and 10% v/v of methanol the differential pulse voltammograms of trans-cinnamaldehyde (−1.05 V) and benzaldehyde (−1.31 V) display reproducible peaks. Under these conditions a strict linearity between both analyte concentrations and their peaks height were observed. The detection limits were calculated to be 2.5 × 10−8 and 1.2 × 10−8 M for trans-cinnamaldehyde and benzaldehyde in pH = 8.2, respectively. The relative standard deviations for 1.0 × 10−6 M of both analytes were 2.04 and 1.18. The heat stability of trans-cinnamaldehyde was studied, and it was found that trans-cinnamaldehyde undergoes a heat-induced decomposition at low temperature (>70°C) to produce benzaldehyde.

Comparative Study of Activity and Heat Stability of Limit Dextrinase in 16 Barley Cultivars

ABSTRACTWe conducted a comprehensive study of 15 cultivars of Chinese barley and one cultivar of European barley to examine and compare the activity and heat stability of limit dextrinase (LD) among them. Our results revealed that the enzymatic activity of LD drops dramatically at incubation temperatures >65°C, indicating that 65°C is the point of incubation temperature sensitivity for this enzyme. Compared with the European barley cultivar Triumph, the levels of enzymatic activity and heat stability of LD in most Chinese barley cultivars included in this study were low, except for Jipi 2 and 92001, which were nearly equal to Triumph. We also found that the activity and heat stability of LD were strongly influenced by the genotype and growth environment of the cultivar, which suggests that genetic manipulation and optimal cultivation conditions may be useful in producing high‐quality barley for the brewing industry.

RF magnetron sputtered ZnO:Al thin films on glass substrates: A study of damp heat stability on their optical and electrical properties

ZnO:Al thin films were deposited on glass substrates by RF magnetron sputtering from a powder compacted ceramic target. Structural, electrical and optical properties of the films with different thickness were characterized. The damp heat stability of ZnO:Al thin film was investigated for its application in thin-film solar cells. After the 1000 h damp heat treatment in harsh conditions of 85% relative humidity at 85 °C for all samples, a degradation of electrical properties was observed, while the transmissions of the films were almost unchanged. Thick films with a relative large grain size could form compact structure to resist the corrosion by oxygen and water molecules.

Fat-free Albumin as a Novel Drug Delivery System

The search for effective drug delivery systems is one of the major challenges in drug formulation especially for biopharmaceuticals such as proteins, and peptide-based drugs and vaccines. A procedure has been developed whereby human serum albumin (HSA) can be used as a delivery vehicle for these biomolecules using its role as main fatty acid carrier. Using essentially fatty acid free HSA (HSAff) it is possible to form stable complexes with lipidic chain compounds (lipo-compounds). Two lipo-compounds have been used to develop this system, a novel antimicrobial lipopeptide and c-amino-n-butyric acid, GABA, conjugated with an alkyl chain, lipo-GABA, in both cases C8 and C14 alkyl chain lengths were evaluated. The HAS–lipo compound complex had a mutual stabilizing effect on both the HSA and the lipo-compound. The protease enzyme study showed that the alkyl chains of these lipocompounds bound to HSAff confer a similar if not greater biostability than caprylic acid shown by CD and importantly, the bound lipopeptide was stabilized by the HSA shown by mass spectrometry. Heat stability studies at 60C over 10 h also confirmed that the lipo-HSA complexes confer stability and provide a method of preparing sterile formulation for therapeutic use. No further increased in stability of the lipo-compounds when HSA containing fatty acid (HSAfa) was used. With the antimicrobial lipopeptide, there was enhanced activity with HSAff formulation suggesting increased biostability and bioavailability of compounds. These finding allowed us to develop a simple and effective way of delivering lipo-compounds using fatty acid free HSA as the carrier.

Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen

Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.

RiV Particles are Heat Stable

The “reaction pattern in vertebrate cells” (RiV) is mainly represented by characteristic particles with a diameter of 50 ± 15 nm and smaller RiV-specific units probably as reaction products of the cells to specific stress. As vaccines they have been successfully applied against laryngeal papillomatosis and experimentally against foot-and-mouth disease of guinea pigs and mammary carcinoma of mice. Subsequently, the small-scale treatment of AIDS, cancer and hepatitis C was started in a single doctor's practice. RiV samples of calf kidney cell culture contain identified proteins and non-viral ribonucleic acid. Vaccine samples and samples for heat stability studies were produced from roller bottle cultivated cells after differential centrifugation and a short solvent treatment. The preparations were examined by electron microscopy, ELISA, protein determination, SDS-PAGE, immunoblot and UV spectroscopy. Since the efficacy of heated RiV (10 min at 100 °C) against the growth of balb/c mouse myeloma was higher than that of the non-heated sample, heated RiV samples were investigated by electron microscopy, immunoelectron microscopy (IEM) and ELISA to find a possible explanation for these results. The RiV particles were surprisingly heat-stable, after 10 (up to 60) min of boiling no differences were observed in morphology and diameter in comparison to the untreated sample. Furthermore, no difference was seen by IEM in the reaction of these RiV particles with an anti-RiV antiserum relative to the untreated sample. When two samples were compared by ELISA using the same antiserum, the reactivity of the heated samples decreased quickly during the first minutes of boiling, but the decrease became much slower later on. In conclusion, there seemed to be heat-sensitive as well as heat-stable reactive components within the RiV samples. The heat-stable component(s) should be associated with the RiV particles. Further studies are necessary to find out whether the positive results of the mouse myeloma experiment could also be obtained following the treatment of other cancers, AIDS and hepatitis C and also to answer the question, which of the known proteins are important for the therapeutic activities of RiV samples.