Determination of human immunodeficiency virus-1 protease inhibitors in patient serum using free solution capillary zone electrophoresis.

The goal of this study was to develop a fast, inexpensive and quantitative method for serum determination of the human immunodeficiency virus protease inhibitors Crixivan (C), Viracept (V), Invirase (I) or Fortovase (F), and Norvir (N), using common conditions for isolation and analysis. The best separation procedure developed thus far involves uncoated silica capillary and a buffer containing formic acid and acetonitrile. This procedure allows us to analyze three drugs (C, V and I or F) in 15 min. Norvir requires different analytical conditions. These four drugs are isolated from patient sera with a mixture of ethyl acetate and hexane. Sensitivity of the capillary zone electrophoresis protocols is sufficient for the detection of these pharmacological agents at the lowest clinically relevant concentrations (0.1 microgram/ml).

A sticker-based model for DNA computation.

We introduce a new model of molecular computation that we call the sticker model. Like many previous proposals it makes use of DNA strands as the physical substrate in which information is represented and of separation by hybridization as a central mechanism. However, unlike previous models, the stickers model has a random access memory that requires no strand extension and uses no enzymes; also (at least in theory), its materials are reusable. The paper describes computation under the stickers model and discusses possible means for physically implementing each operation. Finally, we go on to propose a specific machine architecture for implementing the stickers model as a microprocessor-controlled parallel robotic workstation. In the course of this development a number of previous general concerns about molecular computation (Smith, 1996; Hartmanis, 1995; Linial et al., 1995) are addressed. First, it is clear that general-purpose algorithms can be implemented by DNA-based computers, potentially solving a wide class of search problems. Second, we find that there are challenging problems, for which only modest volumes of DNA should suffice. Third, we demonstrate that the formation and breaking of covalent bonds is not intrinsic to DNA-based computation. Fourth, we show that a single essential biotechnology, sequence-specific separation, suffices for constructing a general-purpose molecular computer. Concerns about errors in this separation operation and means to reduce them are addressed elsewhere (Karp et al., 1995; Roweis and Winfree, 1999). Despite these encouraging theoretical advances, we emphasize that substantial engineering challenges remain at almost all stages and that the ultimate success or failure of DNA computing will certainly depend on whether these challenges can be met in laboratory investigations.

Virologic, immunologic, and clinical evaluation of human immunodeficiency virus antibody status of symptom-free children born to infected mothers.

STUDY OBJECTIVE: To determine the prevalence of infection by the human immunodeficiency virus (HIV) in a population of symptom-free children who were born to HIV-infected mothers and who subsequently underwent seroreversion from an HIV antibody-positive to an HIV antibody-negative status. DESIGN: Cohort. SETTING: Pediatric HIV program in a community setting. PATIENTS: We used HIV DNA polymerase chain reaction (PCR) and coculture to detect the presence or absence of HIV in peripheral blood mononuclear cells of 134 children aged 6 to 53 months. All children had HIV antibody at birth and underwent a subsequent seroreversion to antibody-negative status. RESULTS: In 134 children with HIV antibody-negative status, 219 of 220 culture results and 242 of 247 HIV-1 DNA PCR assay results were negative. Six positive laboratory results were obtained for six different children, each of whom had negative results on multiple assays. For HIV-infected children, 56 of 62 cultures and 99 of 104 PCR evaluations showed positive results. There was no clinical or laboratory evidence of HIV infection in the group with HIV antibody-negative status. CONCLUSION: We were unable to find evidence of latent HIV type 1 infection in this cohort of symptom-free children who underwent seroreversion to HIV antibody-negative status. The loss of maternal HIV antibody in these children indicates the absence of HIV infection. False-positive PCR and culture results occurred sporadically, indicating that repeated analysis of HIV seropositivity in infants and children is necessary.

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.

Virus burden in human immunodeficiency virus type 1-infected children: relationship to disease status and effect of antiviral therapy.

The human immunodeficiency virus type 1 (HIV-1) was isolated from the plasma and peripheral blood mononuclear cells (PBMCs) from each of 21 infected children. The mean titers in plasma were 7 and 165 tissue culture-infective doses per milliliter in 9 children with asymptomatic (P-1) and 12 with symptomatic (P-2) infection, respectively (P = .0013). Significantly higher viral titers were found in PBMCs obtained from P-2 compared with P-1 children: 1920 vs 25 tissue culture-infective doses per 10(6) PBMC (P = .0018). In symptomatic patients at least 1 in 520 circulating mononuclear cells harbored HIV-1. No correlation was found between the viral burden and CD4+ lymphocyte counts. A decrease in the HIV-1 titers was noted both in PBMCs and plasma of symptomatic patients treated with zidovudine for 2 to 7 months. It is concluded that symptomatic children harbor a higher amount of the virus in plasma and PBMCs than asymptomatic children. Zidovudine treatment for 2 months or more decreased the amount of HIV-1 in PBMCs and plasma.

Antigenic properties of vaccinia virus and of the virus recombinant strains expressing heterologous genes.

Immunologic properties of vaccinia virus (VV), strain LIPV, and VV recombinant strains containing the gene of hepatitis B virus surface antigen and the TK gene of herpes simplex virus (HSV) have been studied. Production of antibodies against the majority of VV structural proteins, including nucleocapsid internal proteins was demonstrated in rabbits. Insertion of heterologous genes into the VV genome was without effect on the spectrum of antibodies produced against VV virion proteins. The data obtained in volunteers indicate that not only virus-neutralizing antibodies but also antibodies against most VV structural proteins are preserved in humans over many years. Reimmunization of volunteers with VV recombinant stimulates synthesis of antibodies against virion proteins whereas the spectrum of antibodies remains unchanged. Humans and rabbits did not differ in the spectrum of antibodies to VV virion proteins.

Biologic properties and genome structure of the recombinants between ectromelia and rabbitpox viruses.

Administration of rabbitpox virus (RPV) DNA, cleaved into 2 fragments by SmaI restrictase, into ectromelia virus (EMV)-infected chick fibroblast cells yielded recombinants whose properties were characteristic of both parents. Some recombinants capable of producing RPV-type lesions upon intracutaneous (i.c.) infection of rabbits could also produce EMV-specific lesions upon footpad inoculation of mice. The analysis of some recombinants as well as vaccinia virus strains has shown that the ability of the virus to reproduce when injected into the mouse footpad is a necessary, but not a sufficient condition for production of EMV-type lesions. According to restrictase analysis of recombinant DNA, the genome of recombinants mainly consists of RPV DNA sequences with insertions of small EMV DNA fragments.

Biochemical and genetic characterization of vaccinia virus temperature-sensitive mutants with DNA- and DNAf-phenotypes.

Eighty temperature-sensitive (ts) mutants of vaccinia virus were examined for defects in synthesis of DNA. Nine ts mutants were incapable of synthesizing DNA at the restrictive temperature of 39.5 degrees C (DNA- mutants). Biochemical and genetic data indicate that all 9 DNA- mutants carry mutations in different genes. Temperature shift-up experiments have shown that 6 ts mutants with the DNA- phenotype have mutations in the genes coding for the proteins directly associated with vaccinia DNA synthesis. Temperature shift-down experiments in the presence of cytosine arabinoside revealed 5 ts mutants capable of synthesizing DNA at the elevated temperature, but this DNA failed to form infectious virions. These ts mutants were designated as DNAf- mutants. Pulse-chase experiments for the DNAf- mutant 1877 revealed that viral DNA produced at 39.5 degrees C was incapable of entering into mature virions or any subviral particles. Based on the data for recombination among ts mutants with the DNA- and DNAf- phenotype a tentative genetic map was constructed.

Replication of poxvirus DNA in chick embryo fibroblasts.

Replication of vaccinia virus DNA in chick embryo fibroblasts at a high multiplicity of infection started after 4-6 hr, reached a maximum at the 12th hr and was completed 18 hr after inoculation (p.i.). An analysis of newly synthesised DNAs in the period of the highest rate of synthesis revealed the formation of short (10-30S) fragments which within 30 min were transformed into single-stranded molecules with a sedimentation coefficient of 62S. The final stage of closing of the ends of single-stranded molecules needed much more time and the bulk of "mature" single-stranded 90 S DNA molecules was observed 17 hr p.i.

Tick-borne encephalitis virus-specified sequences in persistently infected cell culture revealed by DNA-DNA hybridization.

Hybridization of DNA probe, obtained through DNA polymerase-mediated in vitro transcription of tick-borne encephalitis virus (TBEV) RNA, with DNA isolated from persistently infected with TBEV cell culture revealed 5.4 copies of viral genome per haploid set.

A comparative study of tick-borne encephalitis virus RNA synthesis in acutely and persistently infected cells.

Rate zonal and buoyant density gradient centrifugation did not reveal any difference between tick-borne encephalitis virus virions released from acutely or persistently infected cells. All three RNA species characteristic for flavivirus replication were found both in acutely or persistently infected cells, but increased levels of intracellular 42S RNA polyadenylation was observed in persistently infected cells.