The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

A wavelet-based adaptive technique for adsorption problems involving steep gradients

A general, fast wavelet-based adaptive collocation method is formulated for heat and mass transfer problems involving a steep moving profile of the dependent variable. The technique of grid adaptation is based on sparse point representation (SPR). The method is applied and tested for the case of a gas–solid non-catalytic reaction in a porous solid at high Thiele modulus. Accurate and convergent steep profiles are obtained for Thiele modulus as large as 100 for the case of slab and found to match the analytical solution.

Computationally efficient solution techniques for adsorption problems involving steep gradients in bidisperse particles

A piecewise uniform fitted mesh method turns out to be sufficient for the solution of a surprisingly wide variety of singularly perturbed problems involving steep gradients. The technique is applied to a model of adsorption in bidisperse solids for which two fitted mesh techniques, a fitted-mesh finite difference method (FMFDM) and fitted mesh collocation method (FMCM) are presented. A combination (FMCMD) of FMCM and the DASSL integration package is found to be most effective in solving the problems. Numerical solutions (FMFDM and FMCMD) were found to match the analytical solution when the adsorption isotherm is linear, even under conditions involving steep gradients for which global collocation fails. In particular, FMCMD is highly efficient for macropore diffusion control or micropore diffusion control. These techniques are simple and there is no limit on the range of the parameters. The techniques can be applied to a variety of adsorption and desorption problems in bidisperse solids with non-linear isotherm and for arbitrary particle geometry.

Adsorption in mesopores: A molecular-continuum model with application to MCM-41

The classical model of surface layering followed by capillary condensation during adsorption in mesopores, is modified here by consideration of the adsorbate solid interaction potential. The new theory accurately predicts the capillary coexistence curve as well as pore criticality, matching that predicted by density functional theory. The model also satisfactorily predicts the isotherm for nitrogen adsorption at 77.4 K on MCM-41 material of various pore sizes, synthesized and characterized in our laboratory, including the multilayer region, using only data on the variation of condensation pressures with pore diameter. The results indicate a minimum mesopore diameter for the surface layering model to hold as 14.1 Å, below which size micropore filling must occur, and a minimum pore diameter for mechanical stability of the hemispherical meniscus during desorption as 34.2 Å. For pores in-between these two sizes reversible condensation is predicted to occur, in accord with the experimental data for nitrogen adsorption on MCM-41 at 77.4 K.

Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus or bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses. (C) 1998 Academic Press.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins. (C) 1997 Elsevier Science B.V.

Adsorption characteristics of carbonized bark for phenol and pentachlorophenol

The potential of using carbonized slash pine bark as a substitute for activated carbon was examined in this study. The bark was carbonized by slow heating in nitrogen for 6.5 h to 672 degrees C. The BET-N-2 surface area, average micropore and mesopore diameter, and micropore volume were 332 m(2) g(-1) 21.7 Angstrom, and 0.125 cm(3) g(-1), respectively. The adsorption capacities for phenol and pentachlorophenol (PCP) at pH 2 and pH 8 were evaluated. The Langmuir equation provided a slightly better fit than the Freundlich equation to two sets of phenol data. The calculated Freundlich constants, K = 0.41 - 0.58 mmol/g/(mmol dm(-3))(1/n) and 1/n = 0.30 - 0.41, were lower and higher, respectively, than literature values for activated carbons. The adsorption capacity of the carbonized bark was much lower for PCP than for phenol. The protonated and anionic PCP isotherms were Type II or III, respectively, in the Brunauer classification. The BET equation provided the best fit to protonated PCP isotherm data. The anionic PCP data were fitted to both the BET model and an equation used in the literature to represent phosphate adsorption on activated carbons. Nonlinear regression of the data for both phenol and PCP adsorption with the Freundlich, Langmuir and BET equations generally gave more accurate parameters, compared with the use of linearized equations to obtain the parameters. (C) 1998 SCI.

Modification of MCM-41 by surface silylation with trimethylchlorosilane and adsorption study

Siliceous MCM-41 samples were modified by silylation using trimethylchlorosilane (TMCS). The surface coverage of functional groups was studied systematically in this work. The role of surface silanol groups during modification was evaluated using techniques of FTIR and Si-29 CP/MAS NMR. Adsorption of water and benzene on samples of various hydrophobicities was measured and compared. It was found that the maximum degree of surface attachments of trimethylsilyl (TMS) groups was about 85%, corresponding to the density of TMS groups of 1.9 per nm(2). The degree of silylation is found to linearly increase with increasing pre-outgassing temperature prior to silylation. A few types of silanol groups exist on MCM-41 surfaces, among which both free and geminal ones are responsible for active silylation. Results of water adsorption show that aluminosilicate MCM-41 materials are more or less hydrophilic, giving a type IV isotherm, similar to that of nitrogen adsorption, whereas siliceous MCM-41 are hydrophobic, exhibiting a type V adsorption isotherm. The fully silylated Si-MCM-41 samples are more hydrophobic giving a type III adsorption isotherm. Benzene adsorption on all MCM-41 samples shows type IV isotherms regardless of the surface chemistry. Capillary condensation occurs at a higher relative pressure for the silylated MCM-41 than that for the unsilylated sample, though the pore diameter was found reduced markedly by silylation. This is thought attributed to the diffusion constriction posed by the attached TMS groups. The results show that the surface chemistry plays an important role in water adsorption, whereas benzene adsorption is predominantly determined by the pore geometry of MCM-41.

Experimental and theoretical investigations of adsorption hysteresis and criticality in MCM-41: Studies with O-2, Ar, and CO2

MCM-41 materials of six different pore diameters were prepared and characterized using X-ray diffraction, transmission electron microscopy, helium pycnometry, small-angle neutron scattering, and gas adsorption (argon at 77.4 and 87.4 K, nitrogen and oxygen at 77.4 K, and carbon dioxide at 194.6 K). A recent molecular continuum model of the authors, previously used for adsorption of nitrogen at 77.4 K, was applied here for adsorption of argon, oxygen, and carbon dioxide. While model predictions of single-pore adsorption isotherms for argon and oxygen are in satisfactory agreement with experimental data, significant deviation was found for carbon dioxide, most likely due to its high quadrupole moment. Predictions of critical pore diameter, below which reversible condensation occurs: were possible by the model and found to be consistent with experimental estimates, for the adsorption of the various gases. On the other hand, existing models such as the Barrett-Joyner-Halenda (BJH), Saito-Foley, and Dubinin-Astakhov models were found to be inadequate, either predicting an incorrect pore diameter or not correlating the isotherms adequately. The wall structure of MCM-41 appears to be close to that of amorphous silica, as inferred from our skeletal density measurements.

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

Solution of adsorption problems involving steep moving profiles

The moving finite element collocation method proposed by Kill et al. (1995) Chem. Engng Sci. 51 (4), 2793-2799 for solution of problems with steep gradients is further developed to solve transient problems arising in the field of adsorption. The technique is applied to a model of adsorption in solids with bidisperse pore structures. Numerical solutions were found to match the analytical solution when it exists (i.e. when the adsorption isotherm is linear). The method is simple yet sufficiently accurate for use in adsorption problems, where global collocation methods fail. (C) 1998 Elsevier Science Ltd. All rights reserved.