DNA microarrays in drug discovery and development.

DNA microarrays can be used to measure the expression patterns of thousands of genes in parallel, generating clues to gene function that can help to identify appropriate targets for therapeutic intervention. They can also be used to monitor changes in gene expression in response to drug treatments. Here, we discuss the different ways in which microarray analysis is likely to affect drug discovery.

Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat.

Tat, the transactivation factor of human immunodeficiency virus type 1 (HIV-1), contains the highly conserved tripeptide sequence Arg-Gly-Asp (RGD) that characterizes sites for integrin-mediated cell adhesion. The tat protein was assayed for cell attachment activity by measuring the adhesion of monocytic, T lymphocytic, and skeletal muscle-derived cell lines to tat-coated substratum. All cell lines tested bound to tat in a dose-dependent manner and the tat cell adhesion required the RGD sequence because tat mutants constructed to contain an RGE or KGE tripeptide sequence did not mediate efficient cell adhesion. The tat-mediated cell attachment also required divalent cations and an intact cytoskeleton. In addition, cell adhesion to tat was inhibited in the presence of an RGD-containing peptide GRGDSPK or an anti-tat mAb that recognizes the RGD epitope. These results strongly suggest that cells are bound to tat through an integrin. Interestingly, myoblast cells bound to tat remained round, whereas the same cells attached through an integrin for a matrix protein typically flatten and spread. The role of this RGD-dependent cellular adhesion of tat in HIV-1 infection remains to be determined.

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.

The 80's loop (residues 78 to 85) is important for the differential activity of retroviral proteases.

The abundance of structural data available for retroviral proteases affords a unique opportunity to investigate structure activity relationships. Our approach attempts to genetically engineer an HIV (human immunodeficiency virus)-1 protease that is functionally equivalent to the HIV-2 and the SIV (simian immunodeficiency virus) enzymes and conversely to engineer an HIV-2 protease that is functionally equivalent to the HIV-1 enzyme. For this purpose, the HIV-2 and SIV proteases were cloned and characterized in an Escherichia coli (E. coli) assay system along with 33 engineered HIV-1 and HIV-2 enzymes. The results of these experiments show that a relatively large S1 or S1' subsite volume, which is likely determined by the conformation of the 80's loop (residues 78 to 85), is necessary to fully accommodate the HIV-1 protease specificity site AETF*YCDG (the asterisk indicates the location scissile bond) during productive binding.

Mutations that alter the DNA binding site for the bacteriophage lambda cII protein and affect the translation efficiency of the cII gene.

The efficiency of translation of the cII gene of bacteriophage lambda is greatly reduced by the cII3059 mutation, a GUU----GAU (Val----Asp) change in the second cII codon. Mutations in the third and fourth codons of the cII gene, called ctr mutations, reverse this translation deficiency. Lambda cII3059 ctr-1, which has a GCA----ACA (Ala----Thr) change in the fourth cII codon, produces about half the normal level of cII activity in liquid cultures, and lambda cII3059 ctr-2 and lambda cII3059 ctr-3, which have identical CGT----CGC changes in the third codon, produce normal levels of cII activity in liquid culture. Since the cII protein of ctr-3 has the same primary sequence as that of lambda cII3059, the cII- phenotype of lambda cII3059 can be explained entirely by the deficiency of translating cII mRNA. We propose that ctr mutations increase translation efficiency by destabilizing a stable stem structure which can be formed by cII mRNA. The ctr mutations lie in an overlapping regulatory region which contains, in addition to sequence elements that influence the rate of cII translation, a region to which cII protein binds to activate transcription from the PRE promoter. The ctr-1 mutation alters the cII recognition sequence from 5'-T-T-G-C-N6T-T-G-C-3' to 5'-T-T-G-C-N6T-T-G-T-3', but has no effect on PRE activity. Since a C----T change in the first (5'-proximal) T-T-G-C sequence (to yield 5'-T-T-G-T-N6T-T-G-C) greatly lowers cII binding affinity, cII protein must not recognize the two T-T-G-C sequences in an identical manner.

Low number of functionally active B lymphocytes in the peripheral blood of HIV-1-seropositive individuals with low p24-specific serum antibody titers.

The in vitro synthesis of HIV-1, p24-, reverse transcriptase (RT)- and gp120-specific immunoglobulin (Ig) G by unstimulated peripheral blood mononuclear cells (PBMC) from 38 asymptomatic and 10 symptomatic HIV-1-seropositive individuals was analysed. In the majority of these individuals, spontaneous production of HIV-1- and gp120-specific IgG from PBMC cultures was demonstrated. In addition, in the majority of the PBMC cultures from individuals with high serum antibody titers to p24, spontaneous production of p24-specific IgG was shown. In contrast, no p24-specific IgG production was detected in PBMC cultures from seropositive individuals with low or no serum antibody titers to p24. A similar relationship between low or absent RT-specific serum antibody titers and the absence of in vitro RT-specific IgG synthesis was not demonstrated. Furthermore, it was shown that the number of p24-specific B lymphocytes in circulation, as calculated by a spot enzyme-linked immunosorbent assay, were significantly lower in individuals with low serum antibody titers to p24. These results suggest that the decline in p24-specific serum antibodies observed during progression towards AIDS is not merely a reflection of the clearance via immune complexes, but may also be attributable, at least in part, to a reduction of p24-specific antibody-producing active B lymphocytes.

Antibody response to the viral negative factor (nef) in HIV-1 infection: a correlate of levels of HIV-1 expression.

Antibody responses against the nef gene product of HIV-1 were determined in sequential sera from a longitudinally studied cohort of 194 initially asymptomatic HIV-1-seropositive individuals and 72 individuals who seroconverted for antibodies to HIV-1 structural proteins (gag/env). In the majority of men, nef-specific antibodies, once detected, persisted (67.6%). In some men, nef-specific antibodies were only transiently (6.8%), or intermittently (5.3%), detectable. No nef-specific antibodies were found in the remaining men (20.3%). Nef-specific antibodies were elicited early in infection, but rarely (2/72 men) prior to seroconversion for antibodies to HIV-1 structural proteins. An absent, transient, or intermittent nef-specific antibody response was significantly associated with the absence or disappearance of antibodies to HIV-1 core proteins, with (re)appearance and persistence of HIV-1 core antigen and with the presence of low CD4+ cell numbers, i.e. profiles previously shown to be predictive of rapid disease progression. Although more cases of AIDS and AIDS-related disease (21/86 versus 28/180) occurred in the nef-specific antibody-negative group than in the nef-specific antibody-positive group, this difference did not reach significance.

Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization.

Cathepsin K is a cysteine protease expressed predominantly in osteoclasts. Activated cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Mutations in the human cathepsin K gene have been demonstrated to be associated with a rare skeletal dysplasia, pycnodysostosis. The degree of functional activity of the mutated forms of cathepsin K in these individuals has not been elucidated, but is predicted to be low or absent. To study the role of cathepsin K in bone resorption, we have generated mice deficient in the cathepsin K gene. Histologic and radiographic analysis of the mice revealed osteopetrosis of the long bones and vertebrae, and abnormal joint morphology. X-ray microcomputerized tomography images allowed quantitation of the increase in bone volume, trabecular thickness, and trabecular number in both the primary spongiosa and the metaphysis of the proximal tibiae. Not all bones were similarly affected. Chondrocyte differentiation was normal. The mice also had abnormalities in hematopoietic compartments, particularly decreased bone marrow cellularity and splenomegaly. The heterozygous animals appeared normal. Close histologic examination of bone histology revealed fully differentiated osteoclasts apposed to small regions of demineralized bone. This strongly suggests that cathepsin K-deficient osteoclasts are capable of demineralizing the extracellular matrix but are unable to adequately remove the demineralized bone. This is entirely consistent with the proposed function of cathepsin K as a matrix-degrading proteinase in bone resorption.

A single vector cloning, mutagenesis and expression system.

We describe a multipurpose Escherichia coli vector, pOTSf1blue, that can be utilized for high efficiency subcloning, epitope-tagged protein overexpression, authentic protein overexpression and efficient mutagenesis.

Regulation of the yeast metallothionein gene.

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.

Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals.

Antibodies to E. coli-produced HIV-1 vpr and vpu were determined by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 196 initially symptom-free men who were positive for such antibodies at study entry. First detection of vpr- and vpu-specific antibodies always was within 12 months of seroconversion for antibodies to structural proteins. In the combined cohort of 268 men, vpr- and vpu-specific antibodies were found persistently in 26 and 43% of men, respectively. Vpr- and vpu-specific antibodies were transiently detected in 3 and 7%, respectively, and intermittently detected in 18 and 13% of men, respectively. No association was found between the patterns of vpr- or vpu-specific antibody response and clinical outcome. In subjects with different patterns of vpr- and vpu-specific antibody response, no clear temporal relationship existed between the appearance or disappearance of antibodies and the onset of HIV-1-related disease.

Contribution of antibody response to recombinant HIV-1 gene-encoded products nef, rev, tat, and protease in predicting development of AIDS in HIV-1-infected individuals.

The relation between antibody-response profiles to Escherichia coli-produced HIV-1 nef, rev, tat, and protease proteins and the risk of developing AIDS was studied using stored serum samples taken sequentially from a cohort of 195 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. The AIDS attack rates at 39 months follow-up were significantly higher in the men with negative versus positive antibody profiles to nef, tat, and protease, respectively. [Difference (D) between attack rates = 11.279, 5.884, and 8.322, respectively]. No significant difference was found between men with negative versus positive antibody profiles to rev. The above differences between AIDS attack rates were clearly lower than those reported from the same cohort for men who were serum HIV-1 antigen positive versus negative, and for men with low versus normal CD4+ lymphocyte counts, but with respect to nef antibody-response profiles, resembled the difference reported between anti-HIV-1 core antibody-negative versus antibody-positive men. In the subgroup of men without any of the markers previously found to be predictive of progression to AIDS in the cohort (persistent HIV-1 p24 antigenemia, low anti-HIV-1 anti-core antibody reactivity, and low CD4+ cell counts), antibody profiles to nef, rev, tat, and protease did not contribute to the prediction of outcome of infection. When used in combination with persistent HIV-1 p24 antigenemia and low CD4+ cell counts, negative antibody profiles to nef and protease, respectively, were equally sensitive and specific in predicting progression to AIDS, as was low anti-HIV-1 anti-core antibody reactivity.

The HIV-1 protease as a therapeutic target for AIDS.

HIV produces a small , dimeric aspartyl protease which specifically cleaves the polyprotein precursors encoding the structural proteins and enzymes of the virus. This proteolytic activity is absolutely required for the production of mature, infectious virions and is therefore an attractive target for therapeutic intervention. This review summarizes the strategies and multidisciplinary efforts that have been applied to date to the identification of specific inhibitors of this critical viral enzyme. These inhibitors include rationally designed peptide substrate analogs, compounds conceived from tertiary structure information on the enzyme and natural products. Future directions in the discovery and development of HIV-1 protease inhibitors are also discussed.

Low antigenicity of HIV-1 rev: rev-specific antibody response of limited value as correlate of rev gene expression and disease progression.

An enzyme immunoassay based on an E. coli-produced HIV-1 rev gene product was used to detect rev-specific antibodies in longitudinally collected serum samples from 196 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. In 61% of men no rev-specific antibodies were detected at all, 30% had persistently detectable rev-specific antibodies, and in 9% rev-specific antibodies were only transiently or intermittently detected. When a persistent rev-specific antibody response occurred in subjects who seroconverted to structural proteins, it was always, with one exception, found within 12 months of seroconversion. The rev-specific antibodies were also studied in a transectional sample of sera from the men who remained symptom-free and from those who developed AIDS-related conditions or AIDS, as well as in sera from 31 other men with AIDS-related conditions and in sera from 6 of these men at the time they developed AIDS. The rev-specific antibodies were found in 34% of symptom-free men, in 28% of patients with AIDS-related conditions, and in 16% of patients with AIDS. The low incidence of rev-specific antibodies early after infection may be due to low antigenicity of rev. The lower prevalence of rev-specific antibodies in sera from patients with AIDS, compared with patients with AIDS-related conditions and symptom-free HIV-1-infected individuals, may be explained by a progressive HIV-1-induced immunodeficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum reactivity to HIV-1 accessory gene products distinguishes East African from West African HIV strains as infecting agent.

PIP: The existence of dual infections with human immunodeficiency virus (HIV)-1 and 2 in West African countries has been controversial, although the current consensus is that dual infection is not the cause of the extensive cross-reactivity observed between these 2 viruses. To evaluate the role of antibody reactivity to the HIV-1 accessory gene products in type-specific HIV serology, proteins encoded for nef, tat, rev, vpr, and vpu were developed and used as an antigen. 5 of the 7 exclusively HIV-2 reactive sera were not reactive to the HIV-1 accessory gene products. Moreover, the 2 sera that showed reactivity to the HIV-1 envelope were the only ones reactive to HIV-1 accessory gene products. These findings indicate that type 2 viruses may be as diverse as type 1 viruses. A subsequent analysis of sera from 24 West Africans revealed reactivity with a simian immunodeficiency virus (SIV) peptide but not with an HIV-1 peptide previously shown to be discriminatory in a direct binding assay between HIV-1 and HIV-2. Compared to 29 control sera from East Africans, the West Africa sera had significantly lower reactivity to antibodies specific to nef, tat, and rev; there was not reactivity to vpr and vpu. 38% of the West African sera compared with 93% of the East African sera showed reactivity to HIV-1 accessory gene products. It is concluded that, while reactivity to the HIV-1 accessory gene products vpr and vpu indicate HIV-1 infection, reactivity to the other accessory gene products cannot be used to identify virus type given the documented cross-reactivity to HIV-1 accessory gene products of antibodies elicited by HIV-2 strains.^ieng

HIV-1 Tat alters normal organization of neurons and astrocytes in primary rodent brain cell cultures: RGD sequence dependence.

The HIV-1 trans-activator protein Tat has been implicated as a mediator of neuronal dysfunction in several model systems. To explore the possibility that Tat can affect primary brain cells, we examined the effect of recombinant Tat protein on rat cortical brain cell cultures. Tat induced marked aggregation of neurons and astrocytes in developing cultures and caused the neuritic processes to coalesce into fascicles. Cell death was not seen and brain macrophages were not affected. These effects mapped to a different region from the trans-activation domain of Tat, as mutating the RGD (arginine-glycine-aspartic acid) sequence within the second exon abrogated aggregation and fascicle formation without affecting trans-activation capacity. Such effects on primary neurons and astrocytes may reflect specific interactions of Tat with uninfected cells within the CNS in vivo.

A human lymphoid recombinant cell line with functional human immunodeficiency virus type 1 envelope.

Our goal has been to develop a safe and effective system that would allow us to explore the functions of the human immunodeficiency virus (HIV) envelope. We have generated a human lymphoid cell line (TF228.1.16) that stably expresses functional HIV envelope proteins on its cell surface, and therefore closely mimics the viral envelope and virus-infected cells. The TF228.1.16 line forms syncytia with human cells of the CD4+ phenotype and provides a facile virus-free cell-based assay for examining the mechanism of syncytia formation and for evaluating novel agents that may disrupt this process. The TF228.1.16 cells also provide an opportunity to present the HIV envelope proteins to the immune system in cellular form. In vitro immunization of human peripheral blood mononuclear cells (PBMC) and in vivo immunization of rhesus monkeys with this reagent results in the production of antibodies with neutralizing (anti-syncytia) activities. When the HIV envelope is expressed against the background of human lymphoid cells, it may exhibit immune protection with unique properties that have not yet been explored. Our results indicate that a virus-free cell system can play an important role in exploring the biology and function of HIV-envelope proteins without the interference of other viral components present in infected cells. This paper discusses these results, and examines the potential use of TF228.1.16 as a vaccine.

[Early exercise test after revascularization and rehabilitation]. / Epreuve d'effort précoce après revascularisation coronaire et revalidation.

Ergospirometry was performed on 51 patients before their discharge from hospital, that is between the seventh and tenth days after myocardial revascularization by cardiac bypass surgery. The aim of our study is to show that this type of measurement can be performed with reasonable safety and that it gives an accurate assessment of the patient's ability to withstand exercise. It employs a metabolic approach: study of oxygen consumption (V'O2), carbon dioxide release (V'CO2), the respiratory quotient (RQ), the minute ventilation (V'E) and the respiratory equivalent for oxygen (REO2). The patients withstood a mean load of 82 +/- 17.7 watts for a mean V'O2 of 1.186 +/- 0.258 l/min STPD and a mean V'E of 46.5 +/- 10 l/min BTPS. Changes in respiratory and metabolic parameters as a function of load are discussed, as is the advice that can be given to the patient regarding physical rehabilitation.