Essential oils for managing Pratylenchus penetrans on Easter lilies.

Easter lily bulbs for greenhouse forcing are produced in Del Norte County, California and Curry County, Oregon, USA. Pratylenchus penetrans infestation seriously affects growth of field grown bulbs. During two consecutive years of field trials containing 22 treatments, commercially prepared formulations of essential oils (EOs) were compared to an untreated control and to a standard chemical fumigant treatment (FU) (1,3-dichloropropene and metam sodium) applied preplant followed by phorate (PH) at planting to determine their value in the management of lesion nematode, and in improving plant health. The EO products Duogard, EF400, EF300, and Cinnamite were tested as preplant dips to bulblet planting stock. The treated bulblets were tested either alone, in combination with PH at-planting, at planting following FU or in combination with PH at planting following FU. The organophosphates ethoprop and fosthiazate were also tested either alone, or at a reduced rate in combination with a reduced rate of PH. With respect to bulb circumference, ten treatments consistently outperformed the control. In consecutive years, three treatments had healthier looking roots than the control. At harvest, levels of lesion nematode within roots were consistently lower in nine treatments. EOs were beneficial in mitigating nematode damage.Easter lily bulbs for greenhouse forcing are produced in Del Norte County, California and Curry County, Oregon, USA. Pratylenchus penetrans infestation seriously affects growth of field grown bulbs. During two consecutive years of field trials containing 22 treatments, commercially prepared formulations of essential oils (EOs) were compared to an untreated control and to a standard chemical fumigant treatment (FU) (1,3-dichloropropene and metam sodium) applied preplant followed by phorate (PH) at planting to determine their value in the management of lesion nematode, and in improving plant health. The EO products Duogard, EF400, EF300, and Cinnamite were tested as preplant dips to bulblet planting stock. The treated bulblets were tested either alone, in combination with PH at-planting, at planting following FU or in combination with PH at planting following FU. The organophosphates ethoprop and fosthiazate were also tested either alone, or at a reduced rate in combination with a reduced rate of PH. With respect to bulb circumference, ten treatments consistently outperformed the control. In consecutive years, three treatments had healthier looking roots than the control. At harvest, levels of lesion nematode within roots were consistently lower in nine treatments. EOs were beneficial in mitigating nematode damage.

A comparative study of the enantiomeric separation of labeled amino acids with cyclodextrins and mixed micelles in capillary electrophoresis.

Enantiomeric separations of fluorescently labeled amino acids are studied by capillary electrophoresis (CE) under a novel variety of experimental conditions. Three different labels are evaluated using two different additives: cyclodextrins (beta- and gamma-) and a dual surfactant system of sodium dodecyl sulfate and sodium taurodeoxycholate. Fluorescein-5-isothiocyanate is the best label to use in this cyclodextrin-based system, and dansyl chloride is the best label to use in this dual surfactant system. Possible limitations for separation of the enantiomers using the mixed micelle system include the fact that there is little interaction of the solute with the surfactants, the negative charge of the solute is limiting the separation window of the system, and the amount of the chiral phase available for partitioning is limited. The separations using cyclodextrins as a chiral selector show that the label affects migration order of the enantiomers, and the cyclodextrins are very effective in separating numerous enantiomers. Overall, cyclodextrins are the better buffer additive for CE use, and the dual surfactant systems, including sodium taurodeoxycholate, offer future promise.

Management Options for Pratylenchus penetrans in Easter Lily.

Alternatives to reduce or modify nematicide use for minimizing groundwater contamination in Easter lily were explored in two field trials. Alternatives to standard 1,3-dichloropropene (1,3-D) plus phorate injection in the first trial were: (i) delaying applications until after winter rains, (ii) removing roots from planting stock, (iii) 1,3-D via drip irrigation, (iv) a chitin-urea soil amendment, (v) the registered insecticide disulfoton, and (vi) several nonregistered nematicides. None of the treatments equaled the standard treatment. In the second trial, potential benefits of adding a systemic nematicide, oxamyl (OX), or a fungicide, metalaxyl (MX), to the standard treatment were explored. Preplant drip irrigation applications of metam sodium (MS), sodium tetrathiocarbonate (ST), and emulsifiable 1,3-D were evaluated alone and in combination with postplant applications of OX and MX. Several drip-applied treatments performed comparably to the standard treatment with respect to the most important criteria of crop quality, bulb circumference. Metam-sodium in combination with either or both OX and MX, 1,3-D plus OX and MX, and ST plus OX and MX provided the best results.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Development of bivalent (B/E) vaccines able to neutralize CCR5-dependent viruses from the United States and Thailand.

Recombinant envelope glycoproteins prepared from a subtype B (MN) strain and a subtype E (CM244) strain of HIV-1 were combined to create a bivalent vaccine (B/E) effective against viruses circulating in the United States and Asia. Combining the two antigens resulted in formulations that increased the breadth and potency of the inter-subtype neutralizing response. Antibodies to the bivalent vaccine formulation neutralized viruses possessing diverse phenotypes, including syncytia-inducing and non-syncytia-inducing primary isolates, viruses using either the CCR5 or the CXCR4 chemokine receptors, and viruses differing in their sensitivity to soluble CD4. These studies demonstrate for the first time that the magnitude and quality of the immune response to HIV-1 can be improved by combining recombinant envelope glycoproteins from different genetic subtypes.

Forced migration and destination choice: Armenian forced settlers and refugees in the Russian Federation.

PIP: The Soviet Republic has experienced major population shifts between its 15 states since 1991. Large numbers of refugees and forced migrants are seeking asylum from successor states in Russia. The Federal Migration Service (FMS) of Russia keeps official registers of refugees and forced settlers, facilitates resettlement, and integrates migrants into society. This study examined the role of the FMS in resettling dislocated persons from Armenia to Russia. Data were obtained from official sources. 49.8% of immigrants to Russia come from the Central Republics. However, Armenia's refugee and forced migrant population is a larger share of its total population (about 5%, compared to 4.4% of Central Asian republics). A 1993 survey revealed that about 70% of the urban populations in Erevan, Gyumri, and Ashtarak would leave Armenia if the opportunity arose. 50% wanted their children to emigrate. In 1989 and 1993, the top receiving areas in Russia were Krasnodarskii Krai, Rostovskaia, and Stavropolskii Krai. In 1989, about 60% of the Armenian population in Russia lived in these territories. Ordinary least squares models indicate that 30% of the variance in Armenian resettlement in Russia, was predicted by high concentrations of Armenian residents and cost of living. Other structural factors, such as unemployment, urbanization, or new construction, were unrelated. Findings suggest that individual choice may be more important in determining residential location than migration policy.^ieng

Problems Associated with Crop Rotation for Management of Pratylenchus penetrans on Easter Lily.

In Humboldt and Del Norte counties of California and Curry County, Oregon, Easter lilies (Lilium longiflotum) are grown commercially in a 3- to 6-year rotation with pasture for cattle and sheep. Bulbs are sold to greenhouse operations to produce flowering plants. The lesion nematode, Pratylenchus penetrans, is a serious detriment to Easter lily production. Both soil and planting stock are often infested; typically, a dual nematicide application is used consisting of a preplant soil fumigation followed by an at-planting application of an organophosphate or carbamate. Nematicide usage has resulted in ground-water contamination. Several factors that could lead to an improved crop rotation program were examined in five field trials in Oregon. Examining the relative nematode host status of crops for feeding cattle and sheep indicated differences in host suitability among clovers and fescues that could prove useful in development of pasture mixes. Populations of P. penetrans under continuous fallow and pasture were monitored for 4 years following harvest of Easter lilies. Populations fluctuated in both situations but generally increased on pasture plants and decreased under fallow. Nematodes were still detectable at the end of 4 years of weed-free fallow. Populations of P. penetrans on Easter lilies were followed over two successive crops. Numbers in soil peaked in July and then decreased while numbers within roots continued to increase until harvest in October.

Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.

Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

sICAM-1 enhances cytokine production stimulated by alloantigen.

The leucocyte adhesion molecules lymphocyte functional antigen-1 (LFA-1; CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1; CD54) facilitate the cell-to-cell interactions which are required for the initiation of immune responses. The role of this interaction in the response to alloantigen was assessed by comparing the effects of monoclonal antibodies against these molecules to the effects of a soluble form of the ICAM-1 molecule in the mixed lymphocyte response (MLR). In contrast to the well-documented inhibitory effects of anti-ICAM-1 or anti-LFA-1 antibodies on mixed lymphocyte responses, we were unable to block these responses with the soluble form of ICAM-1 (sICAM-1). In contrast, the addition of sICAM-1 to these cultures resulted in a two- to sixfold enhancement in the T-cell proliferative response to alloantigen over the normal response. Unlike previous reports, the biological activity of sICAM-1 was not dependent on generation of a solid-phase form of the molecule. The enhanced proliferative response correlated with an increase in the level of TNF-alpha detected in the MLR supernatants and could be blocked by antibodies to TNF-alpha. sICAM-1 had no effect on proliferation or cytokine production in the absence of alloantigen. These results suggest that antibodies which block ICAM-1/LFA-1 not only block the adhesion which is required to stabilize cell-to-cell contact, but also block the costimulatory signal which is required for T-cell activation.

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1).

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) by antisera raised against gp120 from the MN isolate of HIV-1.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.

Resistance of primary isolates of human immunodeficiency virus type 1 to soluble CD4 is independent of CD4-rgp120 binding affinity.

The infection of human cells by laboratory strains of human immunodeficiency virus type 1 (HIV-1) can be blocked readily in vitro by recombinant soluble CD4 and CD4-immunoglobulin hybrid molecules. In contrast, infection by primary isolates of HIV-1 is much less sensitive to blocking in vitro by soluble CD4-based molecules. To investigate the molecular basis for this difference between HIV-1 strains, we isolated the gp120-encoding genes from several CD4-resistant and CD4-sensitive HIV-1 strains and characterized the CD4-binding properties of their recombinant gp120 (rgp120) products. Extensive amino acid sequence variation was found between the gp120 genes of CD4-resistant and CD4-sensitive HIV-1 isolates. However, the CD4-binding affinities of rgp120 from strains with markedly different CD4 sensitivities were essentially the same, and only small differences were observed in the kinetics of CD4 binding. These results suggest that the lower sensitivity of primary HIV-1 isolates to neutralization by CD4-based molecules is not due to lower binding affinity between soluble CD4 and free gp120.

Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160.

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1.

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.

Human immunodeficiency virus type 1 challenge of chimpanzees immunized with recombinant envelope glycoprotein gp120.

The major envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) was purified from a Chinese hamster ovary cell line transfected with a truncated form of the HIV-1 env gene. The recombinant glycoprotein (rgp120) was formulated with aluminum hydroxide adjuvant and was used to immunize chimpanzees. The recombinant preparation was effective in eliciting cellular and humoral immunity as well as immunologic memory. Anti-rgp 120 antibodies reacted with authentic viral gp120 in immunological blot assays and were able to neutralize HIV-1 infectivity in vitro. Sera from the rgp120-immunized animals were able to neutralize HIV-1 pseudotypes of vesicular stomatitis virus prepared from the IIIB isolate, from which the gene encoding rgp120 was derived, as well as two heterologous isolates, ARV-2 and RF. The immune response elicited against the rgp120 was not effective in preventing viral infection after intravenous challenge with HIV-1. The implications of these results on HIV-1 vaccine development are discussed.

Characterization of recombinant human factor VIII.

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.