Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Is there an error correcting code in the base sequence in DNA?

Modern methods of encoding information into digital form include error check digits that are functions of the other information digits. When digital information is transmitted, the values of the error check digits can be computed from the information digits to determine whether the information has been received accurately. These error correcting codes make it possible to detect and correct common errors in transmission. The sequence of bases in DNA is also a digital code consisting of four symbols: A, C, G, and T. Does DNA also contain an error correcting code? Such a code would allow repair enzymes to protect the fidelity of nonreplicating DNA and increase the accuracy of replication. If a linear block error correcting code is present in DNA then some bases would be a linear function of the other bases in each set of bases. We developed an efficient procedure to determine whether such an error correcting code is present in the base sequence. We illustrate the use of this procedure by using it to analyze the lac operon and the gene for cytochrome c. These genes do not appear to contain such a simple error correcting code.     

Ryanodine receptor adaptation and Ca2+(-)induced Ca2+ release-dependent Ca2+ oscillations.

A simplified mechanism that mimics "adaptation" of the ryanodine receptor (RyR) has been developed and its significance for Ca2+(-)induced Ca2+ release and Ca2+ oscillations investigated. For parameters that reproduce experimental data for the RyR from cardiac cells, adaptation of the RyR in combination with sarco/endoplasmic reticulum Ca2+ ATPase Ca2+ pumps in the internal stores can give rise to either low [Cai2+] steady states or Ca2+ oscillations coexisting with unphysiologically high [Cai2+] steady states. In this closed-cell-type model rapid, adaptation-dependent Ca2+ oscillations occur only in limited ranges of parameters. In the presence of Ca2+ influx and efflux from outside the cell (open-cell model) Ca2+ oscillations occur for a wide range of physiological parameter values and have a period that is determined by the rate of Ca2+ refilling of the stores. Although the rate of adaptation of the RyR has a role in determining the shape and the period of the Ca2+ spike, it is not essential for their existence. This is in marked contrast with what is observed for the inositol 1,4,5-trisphosphate receptor for which the biphasic activation and inhibition of its activity by Ca2+ are sufficient to produce oscillations. Results for this model are compared with those based on Ca2+(-)induced Ca2+ release alone in the bullfrog sympathetic neuron. This kinetic model should be suitable for analyzing phenomena associated with "Ca2+ sparks," including their merger into Ca2+ waves in cardiac myocytes.     

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S 1 during the transition. Correspondence to: Y. K. Levine     

Complex Frameshift Mutations Mediated by Plasmid Pkm101: Mutational Mechanisms Deduced from 4-Aminobiphenyl-Induced Mutation Spectra in Salmonella

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in ~2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr(+)) and TA1538 (δuvrB). In strain TA98 (δuvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, ~85% of the revertants also contained the hotspot deletion; the remaining ~15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or + 1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G·C -> T·A transversions) is consistent with the finding that 4-AB induced primarily G·C -> T·A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.