Analysis of Insulin Analogs and the Strategy of Their Further Development

We analyzed the structural properties of the peptide hormone insulin and described the mechanism of its physiological action, as well as effects of insulin in type 1 and 2 diabetes. Recently published data on the development of novel insulin preparations based on combining molecular design and genetic engineering approaches are presented. New strategies for creation of long-acting insulin analogs, the mechanisms of functioning of these analogs and their structure are discussed. Side effects of insulin preparations are described, including amyloidogenesis and possible mitogenic effect. The pathways for development of novel insulin analogs are outlined with regard to the current requirements for therapeutic preparations due to the wider occurrence of diabetes of both types.     

Impact of virus preparation quality on parvovirus filter performance

Virus‐removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus‐removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010 ). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations. Biotechnol. Bioeng. 2013; 110: 229–239. © 2012 Wiley Periodicals, Inc.     

Comparative analysis of the HBsAg level in human blood preparations

A total of 218 batches of blood preparations produced from different raw materials have been studied by means of enzyme immunoassay kits (Abbott Laboratories, USA). The assays have revealed that the preparations under study are nonstandard with respect to the content and isolation rate of HBsAg, the marker of hepatitis B virus. These data necessitate search for the ways of improving the quality of blood preparations.     

Exploiting features of adenovirus replication to support mammalian kinase production

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.     

The use of abrasives in the transmission of plant viruses

The effect of different abrasives on the transmissibility of several plant viruses was tested. Celite and animal charcoal were as effective as carborundum in increasing the number of lesions produced by a given inoculum: 400-mesh carborundum was the most effective among the different sizes which were tested; this gave a result equivalent to increasing the virus content a hundred times. Some preparations of carborundum and charcoal reduced infectivity.
Uninjured plants resisted infectivity when virus solutions were sprayed over them. Leaves previously nibbed without abrasives developed only few lesions whereas leaves rubbed with abrasives developed large numbers. Three hours after rubbing with abrasives leaves had regained their resistance to sprayed virus solutions.
The effects of rubbing leaves with abrasives are described and their significance discussed.     

Effectiveness of the combined use of an inactivated vaccine and a proteolysis inhibitor in the prevention of experimental influenza

The prophylactic effects produced by different types of antiviral preparations, used separately or in combination, in experimental lethal infection induced by influenza virus AO/32 (H0N1) in mice are compared. The use of inactivated vaccine and E-aminocaproic acid (E-ACA), a proteolysis-inhibiting agent, was studied. The qualitative characterization and quantitative evaluation of the anti-influenza effect were carried out by the method of multifactor analysis with the use of a computer after the optimum second-order plan based on the mathematical theory of experiment. This made it possible to determine the best combination of the preparations and their doses, to establish the time of the formation of reliable protection from influenza in mice. The prophylactic effect produced by the use of E-ACA alone and the capacity of this preparation for enhancing the protective action of inactivated influenza vaccine were established. Mathematical analysis revealed the optimum value of the four factors under study: the dose of the vaccine, the dose of E-ACA, the lapse of time between the injection of the preparations and the challenge of the animals, as well as the infective dose of the pathogenic virus. A special experiment made in the study of these data confirmed that the specific formation of a high level of anti-influenza protection in mice can be achieved by the combined use of the vaccine and the inhibitor of proteolysis.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.     

Physico-chemical characteristics of the DNA of the nuclear polyhedrosis virus of the moth Porthetria dispar L

DNA preparations were obtained after dissolving the inclusion bodies, polyhedra virus particles, from the purified bundle virus of Porthetria dispar L. nuclear polyhedrosis. The DNA molecules in the preparations obtained are of different conformation and separate within the CsCl density gradient in the presence of ethidium bromide into supercoiled catenated and relaxed circular molecules (with the admixture of linear molecules). The circular DNA was studied by electron microscopy. The size of virus genome according to the data of reassociation kinetics of DNA is about 100 MD. Estimated on the basis of the values of buoyant density (p) and the melting temperature (Tmelt.) the content of guanine-cytosine pairs (GC pairs) in the viral DNA varies from 61 up to 65 mol%, and in the insect cell DNA--from 38 up to 40 mol%. The viral and cellular DNA are distinctly separated by centrifugation within the CsCl density gradient.     

GSK-3-associated signaling is crucial to virus infection of cells

Signal transduction pathways play important roles in virus infection, replication, and associated pathogenesis. Some of the best understood cell signaling networks are crucial to virus infections such the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and the WNT/β-catenin pathways. Glycogen synthase kinase-3 (GSK-3) is a lesser known signaling molecule in the field of virus research. Interestingly, GSK-3 forms the crux of multiple cell signaling pathways. However, recent studies indicate that GSK-3 may perform key roles in the response to viral infection, replication and pathogenesis. The effects of activated or inactivated forms of GSK-3 on virus infection are still not yet clearly understood phenomenon. The comprehension of the molecular mechanisms underlying the regulation of GSK-3-associated signaling pathways in terms of different stages of virus replication could be important not only to understand the pathogenesis of virus, but also possibly leading to new therapeutic targets. This review will focus on recent advances in understanding the roles of GSK-3 on viral replication, pathogenesis and the immune responses.     

The influence of sodium dodecyl sulfate from different sources on the separation of virus proteins in polyacrylamide gel electrophoresis

Sodium dodecyl sulfate (SDS) preparations from different sources were investigated with regard to their effect on the separation of the proteins of foot and mouth disease virus (FMDV) types 0₁K, A₂S and C-Obb as well as of Sindbis virus during gel electrophoresis in continuous and discontinuous buffer systems in the presence of 8 m urea. In the continuous system, the different SDS preparations did not markedly alter the separations of any of the virus proteins, but the distances between the FMDV protein bands were small. In the discontinuous system, the source of SDS and the kind of virus protein strongly affected the separations obtained. In this system, adequate resolutions were obtained only with A₂S and C-Obb proteins in the presence of MCB or Pierce SDS preparations. An appreciable percentage of the carbon chains in these SDS preparations were found to be longer than C₁₂. The separations of Sindbis virus proteins which occurred in the continuous buffer PAGE system using any of the SDS preparations were preferable to those which were obtained in the discontinuous system.     

Elimination of Mycoplasma Contaminants from Virus Stocks by Treatment with Nonionic Detergents

Five nonionic detergents (Tweens 20, 40, 60, and 80, and Triton WR-1339) were tested for their ability to inactivate four Mycoplasma species which are common contaminants of animal cell cultures. Tween 20 was found to be the most effective, in that a concentration of 2.5 mg/ml completely inactivated cultures of M. hominis, M. hyorhinis, and Acholeplasma laidlawii within 1 hr and a culture of M. orale within 3 hr. The other detergents exhibited various degree of activity against the different mycoplasmas, with Triton WR-1339 being the least effective. The virucidal activity of the detergents was determined for six viruses. All four Tween compounds were highly virucidal for herpes simplex virus. Tween 20 also exhibited virucidal effects against vesicular stomatitis virus, California encephalitis virus, and Newcastle disease virus, and Tween 80 was found to be active against California encephalitis and Newcastle disease viruses. Detergent treatment procedures were effective in two instances in eliminating mycoplasma contaminants from virus preparations while the preparations retained most of the viral infectivity. The limitations of this technique for routine use are discussed.     

Standardization of preparations intended for the serodiagnosis of arboviruses

As the result of our research work, 3 reference preparations have been first obtained and studied in accordance with all requirements of biological standardization. These preparations are the national standard of yellow fever antiserum and immune ascitic fluids (IAF) used as reference reagents: IAF to tick-borne encephalitis virus and IAF to Japanese encephalitis virus. The new preparations are stable, possess sufficient specific activity and can be used as standard preparations for the identification of the above-mentioned viruses.     

Innocuity testing of foot-and-mouth disease vaccines. II. Aziridine-inactivated antigen produced in baby hamster kidney cells

Methods for the testing of preparations of aziridine-inactivated foot-and-mouth disease virus for the absence of infective particles were studied. The system used for virus production, suspension cultures of baby hamster kidney cells, proved to be the most sensitive detection system for traces of infective virus as long as the 146S antigen concentration was below 1 microgram per 10(6) cells. Above this level interference may mask the presence of non-inactivated virus. Thus in a 1-1 suspension culture 1 mg of inactivated 146S antigen equivalent to at least 300 doses of vaccine could be tested. The kinetics of inactivation may be studied by the agar-cell suspension plaque assay which is nearly equal in sensitivity to the method described above. Antigen concentrations at which interference occurred were also estimated for this type of assay. Inactivation of polyethylene glycol-concentrated virus showed 'tailing-off' and such virus preparations should not be used in vaccine production. The data are discussed with reference to the recommendations for innocuity testing in the European Pharmacopoeia.     

Verstärkung der antiphytoviralen Wirkung von 2,4-Dioxohexahydro-l,3,5-triazin durch Kombination mit Verbindungen mit Guanidinstruktur

Intensified antiphytoviral activity of 2,4-dioxohexahydro-l,3,5-triazine by combination with guanidines Combined application of the antiphytoviral compound 2,4-dioxohexahydro-l,3,5 triazine (DHT) and different guanidines (GDs) that were either unsubstituted or substituted only by low-molecular substituents reduced the concentration of potato virus χ (PVX) in leaves or Nicotiana tabacum L. cv. Samsun much more than application of either agent alone. In secondarily PVX-infected leaves, the activity of 2,4-dioxohexahydro- 1,3,5-triazine was increased by a larger number of GDs and to a greater degree than in inoculated ones. The activities of GDs against tobacco mosaic virus (TMV) in Nicotiana tabacum L. cv. Samsun as well as against brome grass mosaic virus (BRV) in Hordeum vulgare L. cv Vogelsanger Gold were only insignificantly increased by combination with DHT. On the other hand, in experiments with so-called identical potato eye cuttings, in which several eye cuttings were obtained from each potato tuber, one serving as a control and the others being treated with DHT, N-cyano guanidine or a combination of these substances, the number of cuttings with symptoms of potato leaf-roll virus (PLRV) could be much more greatly reduced by the combination than by the individual preparations. The number of cuttings with symptoms of potato virus Y and potato virus A was significantly reduced by treatment with the combined preparation, but not by treatment with DHT or N-cyano guanidine alone. Additional investigation with N-cyano-GD, and, beside this, with acetyl-GD and N N′ N triamino-GD indicated a close correlation between the diminution of numbers of potato eye cuttings with virus symptoms and the increase in tuber weight. The greater the reduction in the number of cuttings showing virus symptoms, the greater was the increase in tuber weight. These relationships were observed even in those cases where controls had been treated with ammonium nitrate solutions whose N contents equivalent to the N contents of the preparations. The observed effects of the preparations therefore are not attributable to N-fertilizing effects.     

In Vivo Production, Stabilization, and Infectivity of Baculovirus Preoccluded Virions

Wild-type and polyhedrin-negative isolates of Autographa californica nuclear polyhedrosis virus were replicated in fifth-instar Trichoplusia ni larvae. Insect tissues infected with wild-type virus contained two types of virions that are highly infectious when ingested, those occluded in polyhedra and preoccluded virions. Tissue infected with the polyhedrin-negative virus contained only preoccluded virions. The relative potencies of the two types of infected tissue were determined by dose-mortality bioassays by using the neonate droplet feeding procedure. On a fresh weight basis, preparations of tissues infected with the polyhedrin-negative virus were approximately four times more potent than equivalent preparations of tissue infected with wild-type virus. Approximately half of the observed potency of the wild-type-virus preparations was due to polyhedra, and the remaining activity was due to preoccluded virions present in the tissue. The potency of the polyhedrin-negative preparations was not reduced significantly by lyophilization. The polyhedrin-negative isolate produced about 60% more infectious virus per unit of larval weight than did the wild-type isolate. The ability to produce large amounts of high-potency viral preparations in larvae and the convenience of being able to lyophilize the preparations for long-term storage shows promise for the use of preoccluded virus preparations as biopesticides.     

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Its Metabolite 1-Methyl-4-Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.     

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection.

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.     

Crystalline Structures Associated with the Infection of the Potexvirus Argentine Plantago Virus (APla V)

Abstract An electron microscopy study of Argentine plantago virus (APlaV)-infected plants has revealed that the virus induces crystal-like inclusions in both the cytoplasm and the nucleus of infected cells, in addition to the charcteristic aggregates of virus particles formed by most potexviruses. These crystals are produced in different plants and in tissue at different stage of development and they can be readily detected by leaf dip preparations. We conclude that the crystalline inclusions are virus-specific, and therefore can be regarded as a characteristic feature of APlaV infections.