Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1(US4). We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R(h)) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R(h) of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.     

Quality control of CD4+ T-lymphocyte enumeration: results from the last 9 years of the United Kingdom National External Quality Assessment Scheme for Immune Monitoring (1993-2001)

The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4(+) T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4(+) T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration.     

Precise CD4 T-cell counting using red diode laser excitation: for richer,for poorer

BACKGROUND: Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. METHODS: Diode lasers emitting 10-15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. RESULTS: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R(2) = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. CONCLUSIONS: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology.     

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5

Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.     

Through a glass, darkly: data and uncertainty in the AIDS debate

The HIV/AIDS epidemic is the greatest threat to development in much of Africa. It is already the main cause of death in many countries, especially those in Southern Africa. However there is an absence of solid data on the scale and scope of the disease and how it is evolving. In this article we discuss the data on the epidemic – where it comes from and how it is presented. We note the limitations of the use of antenatal clinic surveys – which provide the bulk of our information.
We then turn to the evidence of impact. The paper shows that the long incubation period between infection and illness means that it takes time for HIV infections to turn into AIDS cases, and AIDS cases to translate into deaths with all the consequences of orphaning, poverty and changing population structures. Furthermore it means that once the HIV prevalence has peaked, AIDS impact will take years to work through – this epidemic is a 'long-wave' event.
The paper is premised on the view that HIV causes AIDS and AIDS causes death. It notes that insufficient and/or unreliable data have allowed leaderships to deny the scope and scale of the problem and that this is unacceptable. However it is incumbent on all to accept the moral responsibility for and the moral consequences of their work, and this includes those who gather, interpret and use the data.     

Seasonal photosynthesis and water relations of juvenile loblolly pine relative to stand density and canopy position

To assess the effects of stand density and canopy environment on tree physiology, we measured gas exchange responses of the same needle age class of 16-year-old loblolly pines ( Pinus taeda L.) in thinned (512 trees ha ^-1) and non-thinned treatment plots (2,863 trees ha ^-1) in central Louisiana. Physiological data were collected in the upper and lower canopy positions on 26 sunny days between July 1996 and June 1997 (one-half of the leaf life span). Mean net photosynthesis was highest (4.3 µmol m ^-2 s ^-1) in the spring and closely corresponded with light intensity in the canopy. Photosynthesis in the winter was nearly 3.0 µmol m ^-2 s ^-1, indicating that loblolly pine enables substantial carbon fixation all year around in the Gulf Coastal Plain region. Mean transpiration and stomatal conductance were highest in the summer and lowest in the winter. With increased light availability after thinning, needle photosynthesis, transpiration and stomatal conductance rose 84, 40 and 23%, respectively, in the lower canopy of the thinned-treatment trees. Light-saturated photosynthetic capacity of the lower canopy needles was 5.2 µmol m ^-2 s ^-1 for the thinned treatment and 4.2 µmol m ^-2 s ^-1 for the non-thinned treatment. It is concluded that thinning-induced light penetration through the canopy enhances physiological activities in the lower canopy foliage of residual trees, and that light availability is the only significant variable for predicting needle-level photosynthesis rates.     

Structure of a human carcinogen-converting enzyme,SULT1A1. Structural and kinetic implications of substrate inhibition

Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, metabolizes and/or bioactivates many endogenous compounds and is implicated in a range of cancers because of its ability to modify diverse promutagen and procarcinogen xenobiotics. The crystal structure of human SULT1A1 reported here is the first sulfotransferase structure complexed with a xenobiotic substrate. An unexpected finding is that the enzyme accommodates not one but two molecules of the xenobiotic model substrate p-nitrophenol in the active site. This result is supported by kinetic data for SULT1A1 that show substrate inhibition for this small xenobiotic. The extended active site of SULT1A1 is consistent with binding of diiodothyronine but cannot easily accommodate beta-estradiol, although both are known substrates. This observation, together with evidence for a disorder-order transition in SULT1A1, suggests that the active site is flexible and can adapt its architecture to accept diverse hydrophobic substrates with varying sizes, shapes and flexibility. Thus the crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.     

Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate

The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162DeltaV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV(SF162P4) virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R(h)) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M(w) of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162DeltaV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162DeltaV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162DeltaV2 Env immunogen in a successful HIV vaccine.