Green fluorescent protein as a reporter of gene expression and protein localization.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

Multivariate models for predicting progression to AIDS and survival in human immunodeficiency virus-infected persons.

Nine hundred thirty persons enrolled in the US Air Force Human Immunodeficiency Virus (HIV) Natural History Study were evaluated with a standard battery of 30 potential surrogate markers of disease progression. A risk score for predicting progression to AIDS was then calculated for each patient in the cohort by using the four highest-ranking variables from multivariate analysis: percentage of CD4 CD29 cells, anergy status, age, and hemoglobin. For predicting survival, beta 2-microglobulin replaced age in the Cox model. Stratification according to the risk score demonstrated that rates of progression to AIDS and survival were significantly different between risk groups (P < .0001). The novel combination of these markers results in extremely accurate risk scores, which may serve as the basis for the development of true surrogate markers of disease progression.

Green fluorescent protein as a marker for gene expression.

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

Delayed-type hypersensitivity skin testing predicts progression to AIDS in HIV-infected patients.

OBJECTIVE: To evaluate the prognostic significance of cutaneous delayed-type hypersensitivity (DTH) skin testing in persons infected with HIV. DESIGN: Cohort study. SETTING: United States Air Force (USAF) Medical Center. PATIENTS: Consecutive sample of 889 HIV-infected USAF personnel or dependents undergoing their first staging evaluation from 1985 through August 1990 in the USAF HIV Natural History Study. MEASUREMENTS: All patients were evaluated with DTH skin testing including purified protein derivative and four control skin test antigens: mumps, candida, tetanus toxoid, and trichophyton. In addition, all patients underwent CD4+ T-cell surface marker determinations. The relation between DTH skin test response at first evaluation and progression to Walter Reed stage 6 (presence of an AIDS-defining opportunistic infection) was evaluated using Kaplan-Meier survival analysis. RESULTS: Patients with more than 400 CD4+ T cells/mm3 are more likely than those having fewer than 400 CD4+ T cells per mm3 to respond to at least one (94% compared with 67%, P < 0.001) or at least two (86% compared with 45%, P < 0.001) DTH skin tests. Mean CD4 counts are lower for anergic compared with nonanergic patients and for patients responding to a single control skin test compared with those responding to two or more skin tests (P < 0.05). The DTH skin test response at first evaluation was also found to predict progression to AIDS; the relative risk at 5 years of follow-up was 2.5 (95% CI, 1.2 to 5.2) for anergy compared with a single positive skin test and 3.0 (CI, 1.4 to 6.2) for a single compared with two or more skin test responses. The DTH skin test response at first evaluation was a predictor of progression (P < 0.001) when controlling for initial CD4 count and Walter Reed stage in a Cox proportional hazards regression analysis. CONCLUSIONS: The DTH skin test response, a functional measure of cellular immunity, is an independent predictor of progression to AIDS in persons with HIV.

Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

Primary structure of the Aequorea victoria green-fluorescent protein.

Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

Comparison of spinal fluid beta 2-microglobulin levels with CD4+ T cell count, in vitro T helper cell function, and spinal fluid IgG parameters in 163 neurologically normal adults infected with the human immunodeficiency virus type 1.

Beta 2-microglobulin levels were measured in the cerebrospinal fluid (CSF) and serum of 163 human immunodeficiency virus-positive (HIV+) persons with normal neurologic physical examinations. None were on antiretroviral therapy. Only 3% had a positive CSF HIV p24 antigen test. The CSF beta 2-microglobulin levels increased as the CD4+ T cell count decreased. Intrathecal production of beta 2-microglobulin was suggested by finding CSF concentrations greater than serum concentrations in 15% of patients. The CSF beta 2-microglobulin levels rose as in vitro T helper cell function deteriorated, independent of CD4+ T cell count. CSF beta 2-microglobulin levels paralleled CSF IgG, IgG index, and IgG synthesis. Higher CSF beta 2-microglobulin levels were found in persons with positive CSF oligoclonal bands. CSF beta 2-microglobulin concentration may serve as a marker for subclinical neurologic damage due to HIV. If this is established, defining the effect of anti-HIV interventions on CSF beta 2-microglobulin would be warranted.

CD4+ monocyte counts in persons with HIV-1 infection: an early increase is followed by a progressive decline.

In this study, we asked whether there is a difference in the number of CD4+ and CD4- peripheral blood monocytes as CD4+ T cells decrease during HIV-mediated immunodeficiency. Monocytes and T cells from 90 HIV-positive and 43 HIV-negative persons were analyzed by flow cytometry. The 90 HIV-positive patients represented the entire spectrum of CD4+ T-cell counts. We report that as CD4+ T cells decrease, the number of CD4+ monocytes decrease in parallel. Moreover, significantly higher CD4+ monocyte counts were observed in persons with early stage HIV disease, i.e., greater than 800 CD4+ T cells/mm3, than in HIV-negative persons with greater than 800 CD4+ T cells/mm3. Potential implications of these findings are discussed.

Hepatitis C antibody in a non-hemophiliac cohort infected with the human immunodeficiency virus.

Development of a serologic test which detects antibody to hepatitis C virus (anti-HCV) allowed us to compare the seroprevalence of hepatitis C and hepatitis B in 493 persons infected with the human immunodeficiency virus (HIV). These persons, none of whom are hemophiliacs, are part of the US Air Force HIV Natural History Study. We found that Hepatitis B core antibody (anti-HBc) was far more prevalent (59%) than anti-HCV (8%). Anti-HBc prevalence was not different between those with and those without anti-HCV, being present in the majority of persons in both groups. In addition, we compared anti-HCV+ and anti-HCV negative persons in terms of syphilis serologies (Reactive Plasma Reagent [RPR] and Fluorescent Treponemal Antibody Absorption [FTA-ABS]), hepatic transaminase levels, and racial composition. In this cohort, we found that anti-HCV+ persons are significantly more likely to have a positive RPR but not FTA-ABS, increased hepatic transaminase levels, and to be Black rather than Caucasian.

X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein.

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.

Burkitt's lymphoma in the femoral triangle of a white american male.

A teenage white male was recently evaluated for a rapidly enlarging mass in the right groin. Biopsy of the mass disclosed Burkitt's lymphoma. Aggressive therapy was instituted, including extirpative surgery, followed by chemotherapy and radiation therapy. Using these combined modalities, it was possible to eliminate the lymphoma. Unfortunately, severe pancytopenia and immunosuppression developed and the patient died of gram-negative sepsis. This disease should be considered in the differential diagnosis of any rapidly enlarging solid tumor in a young patient.