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For a potential function (in one dimension) which evolves from a specified initial form V(i)(x) to a different V(f)(x) asymptotically, we study the evolution, in an overdamped dynamics, of an initial probability density to its final equilibrium. There can be unexpected effects that can arise from the time dependence. We choose a time variation of the form V(x,t) = V(f)(x) + (V(i) - V(f)) e(-lambda t). For a V(f)(x), which is double welled and a V(i)(x) which is simple harmonic, we show that, in particular, if the evolution is adiabatic, this results in a decrease in the Kramers time characteristic of V(f)(x). Thus the time dependence makes diffusion over a barrier more efficient. There can also be interesting resonance effects when V(i)(x) and V(f)(x) are two harmonic potentials displaced with respect to each other that arise from the coincidence of the intrinsic time scale characterizing the potential variation and the Kramers time. Both these features are illustrated through representative examples.

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The human immunodeficiency virus (HIV) fusion inhibitor siamycin I, a 21-residue tricyclic peptide, was identified from a Streptomyces culture by using a cell fusion assay involving cocultivation of HeLa-CD4+ cells and monkey kidney (BSC-1) cells expressing the HIV envelope gp160. Siamycin I is effective against acute HIV type 1 (HIV-1) and HIV-2 infections, with 50% effective doses ranging from 0.05 to 5.7 microM, and the concentration resulting in a 50% decrease in cell viability in the absence of viral infection is 150 microM in CEM-SS cells. Siamycin I inhibits fusion between C8166 cells and CEM-SS cells chronically infected with HIV (50% effective dose of 0.08 microM) but has no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I does not inhibit gp120 binding to CD4 in either gp120- or CD4-based capture enzyme-linked immunosorbent assays. Inhibition of HIV-induced fusion by this compound is reversible, suggesting that siamycin I binds noncovalently. An HIV-1 resistant variant was selected by in vitro passage of virus in the presence of increasing concentrations of siamycin I. Drug susceptibility studies on a chimeric virus containing the envelope gene from the siamycin I-resistant variant indicate that resistance maps to the gp160 gene. Envelope-deficient HIV complemented with gp160 from siamycin I-resistant HIV also displayed a resistant phenotype upon infection of HeLa-CD4-LTR-beta-gal cells. A comparison of the DNA sequences of the envelope genes from the resistant and parent viruses revealed a total of six amino acid changes. Together these results indicate that siamycin I interacts with the HIV envelope protein.

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Development of stavudine resistance was studied using human immunodeficiency virus type 1 isolates from 13 patients treated with stavudine for 18-22 months. Drug sensitivity testing on 11 of these pre- and posttherapy isolates identified only 2 posttreatment isolates with decreased stavudine sensitivity (ED50s < 4-fold higher than the average pretreatment ED50). Genotypic analysis of all 13 pairs of isolates identified multiple mutations in the reverse transcriptase (RT) gene. However, no genetic basis was identified to account for the observed changes in stavudine susceptibility. A recombinant virus containing the entire RT gene of the posttherapy isolate displaying the greatest resistance remained sensitive to stavudine. Five of the stavudine posttreatment isolates developed resistance (9- to 176-fold) to zidovudine, although the relationship between stavudine treatment and the appearance of zidovudine resistance remains unexplained. Analysis of 10 additional pairs of isolates did not confirm this relationship. The low frequency and modest degree of change in stavudine sensitivity following prolonged treatment is very encouraging.

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Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.

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We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line. The 125I-rMIP-1 alpha binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor, with a Kd of approximately 1.5 x 10(-9) M and approximately 1200 binding sites/Con A-stimulated CTLL-R8 cell and a Kd of 0.9 x 10(-9) M and approximately 380 binding sites/RAW 264.7 cell. 125I-rMIP-1 alpha binding was inhibited by unlabeled rMIP-1 alpha in a dose-dependent manner, but not by IL-1 alpha or IL-2. rMIP-1 alpha inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti-rMIP-1 alpha antibodies blocked the growth-inhibitory effect of the rMIP-1 alpha on CTLL-R8 cells.

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Angiotensin-converting enzyme (ACE) is present in endothelial and epithelial cells of various tissues as well as in the circulating plasma. The structural relationship between the cellular and the secreted forms of ACE and the pathways to their biosynthesis have not been determined as yet mainly because of the unavailability of a natural cell line expressing ACE in tissue culture. To circumvent this problem we have permanently transfected a mouse epithelial line with an expression vector containing the recently cloned rabbit testicular ACE cDNA. Clonal derivatives of this line secreted large quantities of enzymatically active ACE. When these cells were cultured in serum-free medium, the only detectable protein in the culture medium was ACE. It has been suggested that a hydrophobic domain near the carboxyl terminus of the enzyme anchors it to the plasma membrane. To test this hypothesis we established cell lines expressing a truncated form of the active enzyme which is missing the putative anchoring domain. Pulse-chase experiments showed that the truncated ACE was secreted from the cells much faster than the native enzyme. Moreover, the secreted form of the native enzyme had a lower molecular weight than the corresponding cellular form. These results are consistent with the hypothesis that the hydrophobic domain is instrumental in keeping the enzyme cell-bound, and secretion is achieved physiologically by removal of this domain from the enzyme by a specific proteolytic cleavage.

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We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.

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2'-5'-oligoadenylate synthetases constitute a multimember family of interferon-inducible enzymes which need double-stranded RNA as an obligatory cofactor. We have isolated cDNA clones for two new murine synthetases. These two clones, 9-2 and 3-9, encoded proteins of 414 and 363 amino acid residues, respectively, out of which the amino terminal 346 residues were almost identical. They were also very similar to the corresponding regions of human synthetases E16 and E18. On the other hand, the carboxyl-terminal 68 residues of clone 9-2 had no homology with the carboxyl-terminal residues of E18. These murine clones had only 67% amino acid identity with the previously isolated murine synthetase clone L3. 9-2 and 3-9 proteins were expressed efficiently by in vitro transcription and translation of cDNA clones containing the synthetase coding regions preceded by the 5'-untranslated region of the vesicular stomatitis virus NS gene. These in vitro synthetized proteins bound to double-stranded RNA and catalyzed the synthesis of 2'-5' oligoadenylates. A nested set of deletion mutants of the 9-2 clone was produced by restriction digestion and polymerase chain reaction. Functional testing of the corresponding truncated proteins revealed that a region between amino acid residues 104 and 158 was necessary for binding to double-stranded RNA and a region between residues 320 and 344 was necessary for enzyme activity. Moreover substitution of the lysine residue at position 333 by arginine did not affect the enzyme activity.

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Bovine phenylethanolamine N-methyltransferase (PNMT) cDNA was inserted into a bovine papilloma virus-based expression vector and used to transfect a mouse C127 cell line. The resultant recombinant bovine PNMT was characterized biochemically and immunochemically. Recombinant bovine PNMT activity, like the native bovine enzyme, was enhanced by phosphate ion in a concentration-dependent manner. Their molecular weights were shown to be identical by Western blot analysis. Antibodies raised against native bovine adrenal PNMT equally immunoprecipitated the activity of the recombinant and native enzymes. In addition, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine PNMT. These antibodies immunostained the recombinant enzyme protein in transfected cells and in their neurite-like processes. In addition, in situ hybridization with the bovine PNMT cDNA probe resulted in a labelling pattern similar to the immunostaining. The recombinant bovine PNMT as the native bovine enzyme exist in multiple-charge forms, but only one form is predominant. Taken together, our results suggest that recombinant bovine PNMT, expressed from bovine PNMT cDNA in a mouse cell line is enzymatically active and shares many common features with native bovine adrenal PNMT.

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We have described earlier a gene cluster, including at least six interferon-activatable genes closely linked to the erythroid alpha spectrin locus and the serum amyloid P-component locus on murine chromosome 1. Here, we report that sequences of three genes from the cluster (the 201, 202, and 204 genes) are very similar in a segment extending from at least 550 nucleotides upstream of the 3' end of the transcription initiation region to beyond the first exon intron border (96% similarity between the 202 and 204 genes and 89% similarity between the 201 and 204 genes). This region contains the following two types of interferon-responsive enhancers: a GA box and a Friedman Stark sequence. The proteins coded for by the 202 gene (51 kDa) and the 204 gene (72 kDa) are hydrophilic. The amino acids have been conserved in the two proteins in 47% of the sequence. Each protein includes two apparently contiguous, approximately 200-amino acid long segments with much sequence similarity (27% in the 202 protein and 34% in the 204 protein). These segments are preceded in the 204 protein only by a segment including four perfect and three imperfect repeats of a 7 amino acid sequence. These and other data suggest that the evolution of the gene cluster involved the duplication of a DNA segment generating a double length transcription unit and subsequent divergence and duplication of this unit giving rise to at least two interferon-activatable genes (the 202 and 204 genes).

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The surface antigen of hepatitis B virus (HBV) has been expressed in a mouse cell line using metallothionein-bovine papilloma virus vectors. Four different recombinant plasmids were used and it was found that the expression level varies significantly from one plasmid to another. Removing the HBV polyadenylation signal from the plasmid drastically reduced the expression level. Providing even a heterologous polyadenylation signal improved the expression level from the reduced one by at least tenfold. The steady-state level of cytoplasmic HBV-specific RNA was much higher when the polyadenylation signal was present. One of the constructs gave a very high expression level and has been characterized further. It was possible to detect any plasmid in the episomal form in the producing clones and all the HBV DNA was found to be integrated in the mouse chromosome at more than one site. The copy number of the plasmid varied greatly between different clones and in the best one it is approximately 150 copies per haploid genome. Cells can be grown continuously in the presence of cadmium chloride without splitting for more than two months and the excreted surface antigen can be harvested during this period. Western blot analysis showed that the antigen contains the pre-S region.

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Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.

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We have studied different conditions of the deionization of formamide with Biorad mixed bed resin AG501-X(D) and find that contrary to the popular usage, drying the resin before deionization produces the best results. In a typical deionization procedure, conductivity initially goes down, reaches a minimum plateau and finally goes up again. The initial rate of deionization, the minimum conductivity reached at the plateau and the rate of final rise in conductivity depend on whether the resin is dry or wet (i.e., straight from the bottle) and on the amount of resin used. In general, wet resin produced faster initial deionization, higher minimum conductivity and quicker final rise in conductivity. Surprisingly, a smaller amount (5%) of resin worked better than a larger amount (20%). With smaller amount of resin, although initial deionization was slower, the minimum conductivity achieved was lower and the final rise in conductivity was slower. This was partly due to the fact that the conductivity of formamide increases faster with increasing amount of water in it.

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Treatment of cells with interferons induces various mRNAs and the corresponding proteins. We have described previously the isolation of a mouse cDNA clone (cDNA clone 202) which specifies an mRNA whose level is increased 20-fold in beta-interferon-treated Ehrlich ascites tumor cells. The increase is a consequence of an increased rate of transcription. The mRNA encodes a 56,000-dalton protein. We report here the isolation of a genomic clone including the 5' terminus of the 202 gene with the interferon-responsive region. Experiments involving primer extension and protection from cleavage by S1 nuclease revealed the existence of multiple 5' termini of 202 mRNAs in Ehrlich ascites tumor and Ltk- cells. Treatment with beta-interferon increased the level of these 202 mRNAs with different 5' termini nonuniformly. A 0.8-kilobase DNA segment from the 202 gene (including its 5' flanking region and its 5'-terminal exon) was ligated to the chloramphenicol acetyltransferase gene, and the resulting construct was transfected into mouse Ltk- cells. Treatment of these cells with beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Within the first, untranslated exon of the 202 gene, we found a 29-nucleotide long sequence that is partially homologous to sequences which occur upstream from interferon-inducible human HLA and metallothionein IIA genes (Friedman, R. L., and Stark, G. R. (1985) Nature 314, 637-639).

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The exposure of cells to interferons enhances the accumulation of particular mRNAs and of the corresponding proteins. A cDNA clone (clone 202) complementary to an mRNA (202 mRNA) whose level is enhanced over 12-fold in mouse Ehrlich ascites tumor cells upon exposure to beta-interferon for 10 hr has previously been isolated. The level of this mRNA was also increased in other beta-interferon-responsive mouse cell lines (i.e., L929, L1210S) but not in a line (L1210R) which is not responsive to beta-interferon. The extent of induction in Ehrlich ascites tumor cells depended on the beta-interferon concentration and reached its maximal level between 300 and 1000 units of interferon/ml. Nuclei isolated from Ehrlich ascites tumor cells which had been exposed to beta-interferon produced in vitro more 202 specific RNA than nuclei from control Ehrlich ascites tumor cells: an increase in this production was detectable 2 hr after beginning the exposure of the cells to 1000 units/ml of beta-interferon and the increase reached its maximal level, around 18-fold, after 18 hr exposure. Much, if not all of this increase, appeared to be due to an increase in the rate of synthesis of the RNA and not to a decrease in its rate of turnover. The 202 mRNA was translated in a reticulocyte lysate into a 56,000-Da protein.

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The 202 gene which specifies a 56.5 kD protein can be induced in Ehrlich ascites tumor cells by treatment with mouse beta interferon. This treatment increases the level of the gene 202-specific mRNA at least 12-fold. For determining the chromosomal location of this gene a 1.5 kb fragment of the gene was hybridized to EcoR1 digested DNA samples from a set of mouse-hamster somatic cell hybrids. Each of the cell hybrids used contained a complete array of hamster chromosomes and one or more mouse chromosomes. The 202 gene fragment hybridized to every DNA sample from cell hybrids containing mouse chromosome 1 (8 hybrids in total) and to none of the DNA samples from hybrids lacking this chromosome (7 hybrids in total). These and other data indicate that the 202 gene is located on mouse chromosome 1.

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(2'-5')(A)n synthetase and RNAase L (a latent endoribonuclease) are among the mediators of interferon action. The product of (2'-5')(A)n synthetase (i.e., (2'-5')(A)n) binds, and thereby activates RNAase L. Interferons induce in Ehrlich ascites tumor (EAT) cells two mRNAs (sizes 1.5 kb and 3.8 kb), which can be translated in Xenopus oocytes into (2'-5')(A)n synthetases of 20,000 to 30,000 daltons and 85,000 to 100,000 daltons, respectively. (2'-5')(A)n synthetases of corresponding sizes are induced by interferons in EAT cells. In the cell extract the bulk of the larger enzyme is in the cytoplasmic fraction, and the bulk of the smaller one in the nuclear fraction. The only known function of (2'-5')(A)n is the activation of RNAase L, and RNAase L can be selectively crosslinked to a (2'-5')(A)n derivative in a cytoplasmic extract from EAT cells. The same (2'-5')(A)n derivative can be crosslinked to several proteins in the nuclear extract of EAT cells, and some of these proteins are induced by interferon.

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(2'-5')(A)n synthetase is one of the mediators of interferon action. If activated by double-stranded RNA it converts ATP into pyrophosphate and (2'-5')(A)n. In turn, (2'-5')(A)n activates a latent endoribonuclease (RNase L) which cleaves single-stranded RNA. We report here the isolation and characterization of a homogeneous human (2'-5')(A)n synthetase. The enzyme was purified from interferon-treated HeLA S3 cells by chromatography of a ribosomal salt wash fraction on DEAE-cellulose, poly(I) . poly(C) agarose, and CM-cellulose. The purified (2'-5')(A)n synthetase can convert over 90% of ATP into (2'-5')(A)n. The enzyme is unstable but can be stabilized by certain nonionic detergents (e.g. Triton X-100). Its apparent Mr = 100,000, as determined by gel electrophoresis in sodium dodecyl sulfate, and about 80,000, as determined by centrifugation through a glycerol gradient. The human (2'-5')(A)n synthetase is similar to the corresponding enzyme from mouse Ehrlich ascites tumor cells, but differs from the latter in size (100,000 versus 105,000 daltons) and in ionic conditions required for maximal activity.

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