Consequences of metal exposure on retinoid metabolism in vertebrates: a review.

What we generally refer to as 'vitamin A' is a group of naturally-occurring molecules structurally similar to retinol that are capable of exerting biological activity. These retinoids are essential to diverse physiological functions including vision, immune response, bone mineralization, reproduction, cell differentiation, and growth. As well, some retinoids have antioxidant properties. Independent studies published over the last few decades have revealed that many fish and wildlife populations living in highly polluted environments have altered retinoid status possibly associated with retinoid metabolic or homeostatic mechanisms. Substantial evidence links organic contaminant exposure with changes in retinoid status in animal populations, but only a few detailed studies have been published implicating inorganic contaminants such as metals. This mini-review selectively deals with field and laboratory studies reporting associations between environmental contaminants, especially trace metals, and alterations in retinoid status. Both essential and non-essential trace metals have been reported to affect retinoid status. This review focuses on metabolic imbalances of retinoids in relation to metal contamination and illustrates possible modes of action. The role of retinoids as antioxidants and their potential as biomarkers of metal contamination are discussed.

Thermally assisted quantum annealing of a 16-qubit problem.

Efforts to develop useful quantum computers have been blocked primarily by environmental noise. Quantum annealing is a scheme of quantum computation that is predicted to be more robust against noise, because despite the thermal environment mixing the system's state in the energy basis, the system partially retains coherence in the computational basis, and hence is able to establish well-defined eigenstates. Here we examine the environment's effect on quantum annealing using 16 qubits of a superconducting quantum processor. For a problem instance with an isolated small-gap anticrossing between the lowest two energy levels, we experimentally demonstrate that, even with annealing times eight orders of magnitude longer than the predicted single-qubit decoherence time, the probabilities of performing a successful computation are similar to those expected for a fully coherent system. Moreover, for the problem studied, we show that quantum annealing can take advantage of a thermal environment to achieve a speedup factor of up to 1,000 over a closed system.

Generation of neutralizing aptamers against herpes simplex virus type 2: potential components of multivalent microbicides.

The prophylactic use of topical antiviral agents has recently been validated by the reduction in human immunodeficiency virus (HIV) type 1 infection incidence seen using tonofovir-containing microbicides. In order to develop a wide-spectrum microbicide to prevent infection with a wide range of sexually transmitted viruses, we have previously reported the development of HIV-neutralizing aptamers and here report the isolation and characterization of aptamers that neutralize herpes simplex virus type 2 (HSV-2). These aptamers bind the envelope glycoprotein (gD), are potent (IC(50) of 20-50 nM) and are able to block infection pathways dependent on both major entry receptors, Nectin1 and HVEM. Structural analysis and mutagenesis of these aptamers reveal a core specificity element that could provide the basis for pharmaceutical development. As HSV-2 is a major risk factor for the acquisition of HIV-1, a microbicide capable of preventing HSV-2 infection would not only reduce the morbidity associated with HSV-2, but also that derived from HIV-1.

Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system.

The molecular mechanisms underlying the directional neuron-to-epithelial cell transport of herpesvirus particles during infection are poorly understood. To study the role of the viral glycoprotein D (gD) in the directional spread of herpes simplex virus (HSV) and pseudorabies virus (PRV) infection, a culture system consisting of sympathetic neurons or epithelial cells in different compartments was employed. We discovered that PRV infection could spread efficiently from neurons to cells and back to neurons in the absence of gD, the viral ligand required for entry of extracellular particles. Unexpectedly, PRV infection can also spread transneuronally via axo-axonal contacts. We show that this form of interaxonal spread between neurons is gD independent and is not mediated by extracellular virions. We also found that unlike PRV gD, HSV-1 gD is required for neuron-to-cell spread of infection. Neither of the host cell gD receptors (HVEM and nectin-1) is required in target primary fibroblasts for neuron-to-cell spread of HSV-1 or PRV infection.

Effects of dietary administration of CL-20 on Japanese quail Coturnix coturnix japonica.

Hexanitrohexaazaisowurtzitane, or CL-20, is an emerging highly energetic compound currently under consideration for military applications. With the anticipated wide use of CL-20, there is the potential for soil and groundwater contamination resulting in adverse toxicologic effects on environmental receptors. Presently, there is a lack of data describing the toxic effects of CL-20 on avian species. The present study describes the effect of CL-20 on Japanese quail (Coturnix coturnix japonica) modified from standard toxicity test guidelines. First, a 14-day subacute assay was adopted using repeated gavage doses (0, 307, 964, 2439, 3475, or 5304 mg CL-20/kg body weight (BW)/d for 5 days followed by no CL-20 exposure (vehicle only) for 10 days. Second, a subchronic feeding assay (0, 11, 114, or 1085 mg CL-20/kg feed) was done for 42 days. During both studies, no overt toxicity was observed in the CL-20-treated birds. During the first 5 days of the subacute study, CL-20-exposed birds showed a dose-dependent decrease in BW gain, whereas increased liver weight, plasma sodium, and creatinine levels were observed in birds receiving the highest dose tested. For the subchronic study, embryo weights were significantly decreased in a dose-dependent manner. Embryos from CL-20-exposed birds were observed to have multiple cranial and facial deformities, beak curvatures, possible mid-brain enlargement, and classic one-sided development with micro-opthalamia (nonstatistical comparisons with control embryos). A trend toward decreased number of eggs laid per female bird was also observed. We conclude that CL-20 (or its degradation products) elicits few effects in adults but may affect avian development, although these preliminary findings should be confirmed.

Advanced electronics for the CTF MEG system.

Development of the CTF MEG system has been advanced with the introduction of a computer processing cluster between the data acquisition electronics and the host computer. The advent of fast processors, memory, and network interfaces has made this innovation feasible for large data streams at high sampling rates. We have implemented tasks including anti-alias filter, sample rate decimation, higher gradient balancing, crosstalk correction, and optional filters with a cluster consisting of 4 dual Intel Xeon processors operating on up to 275 channel MEG systems at 12 kHz sample rate. The architecture is expandable with additional processors to implement advanced processing tasks which may include e.g., continuous head localization/motion correction, optional display filters, coherence calculations, or real time synthetic channels (via beamformer). We also describe an electronics configuration upgrade to provide operator console access to the peripheral interface features such as analog signal and trigger I/O. This allows remote location of the acoustically noisy electronics cabinet and fitting of the cabinet with doors for improved EMI shielding. Finally, we present the latest performance results available for the CTF 275 channel MEG system including an unshielded SEF (median nerve electrical stimulation) measurement enhanced by application of an adaptive beamformer technique (SAM) which allows recognition of the nominal 20-ms response in the unaveraged signal.

Retinoid metabolism (LRAT, REH) in the yolk-sac membrane of Japanese quail eggs and effects of mono-ortho-PCBs.

Retinoids stored in the avian egg are essential for normal development, however, laboratory and field experiments suggest that they are affected by environmental contaminants. Lecithin:retinol acyltransferase (LRAT) activity was detected in the microsomal fraction of the yolk-sac membrane of the Japanese quail at day 6 of development. LRAT activity was maximal at pH 7.0 having apparent kinetic parameters of K(m)=1.35 microM and V(max)=0.21 nmol/mg protein/h and was inhibited by the sulfhydryl modifying agent N-ethyl-maleimide. Retinol ester hydrolase (REH) activity in the microsomal fraction of the yolk-sac membrane was stimulated by the bile salt analogue 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate and was maximal at pH 9.0 with apparent K(m)=77 microM and V(max)=34.3 nmol/mg protein/h. Injection of the PCB congener 2,3,3',4,4'-pentachlorobiphenyl increased both REH and LRAT activities, whereas 2,3,3',4-tetrachlorobiphenyl stimulated LRAT. Yolk retinol concentration and the molar ratio retinol:retinyl palmitate were lower in the exposed eggs. Yolk retinol concentration decreased as LRAT increased (R(2)=0.89) suggesting that certain PCB congeners may affect vitamin A mobilization in ovo by increasing LRAT activity in the yolk-sac membrane.

Structural features of nectin-2 (HveB) required for herpes simplex virus entry.

One step in the process of herpes simplex virus (HSV) entry into cells is the binding of viral glycoprotein D (gD) to a cellular receptor. Human nectin-2 (also known as HveB and Prr2), a member of the immunoglobulin (Ig) superfamily, serves as a gD receptor for the entry of HSV-2, variant forms of HSV-1 that have amino acid substitutions at position 25 or 27 of gD (for example, HSV-1/Rid), and porcine pseudorabies virus (PRV). The gD binding region of nectin-2 is believed to be localized to the N-terminal variable-like (V) Ig domain. In order to identify specific amino acid sequences in nectin-2 that are important for HSV entry activity, chimeric molecules were constructed by exchange of sequences between human nectin-2 and its mouse homolog, mouse nectin-2, which mediates entry of PRV but not HSV-1 or HSV-2. The nectin-2 chimeric molecules were expressed in Chinese hamster ovary cells, which normally lack a gD receptor, and tested for cell surface expression and viral entry activity. As expected, chimeric molecules containing the V domain of human nectin-2 exhibited HSV entry activity. Replacement of either of two small regions in the V domain of mouse nectin-2 with amino acids from the equivalent positions in human nectin-2 (amino acids 75 to 81 or 89) transferred HSV-1/Rid entry activity to mouse nectin-2. The resulting chimeras also exhibited enhanced HSV-2 entry activity and gained the ability to mediate wild-type HSV-1 entry. Replacement of amino acid 89 of human nectin-2 with the corresponding mouse amino acid (M89F) eliminated HSV entry activity. These results identify two different amino acid sequences, predicted to lie adjacent to the C' and C" beta-strands of the V domain, that are critical for HSV entry activity. This region is homologous to the human immunodeficiency virus binding region of CD4 and to the poliovirus binding region of CD155.

Transcription from the gene encoding the herpesvirus entry receptor nectin-1 (HveC) in nervous tissue of adult mouse.

Both human and murine forms of nectin-1 (HveC, Prr1) can serve as entry receptors for several neurotropic herpesviruses, including herpes simplex viruses 1 and 2 (HSV-1, HSV-2), porcine pseudorabies virus (PRV), and bovine herpesvirus 1. HSV-1, HSV-2, and PRV can cause lethal neurological disease in mice whether inoculation is directly into the central nervous system or by peripheral routes. Expression of nectin-1 transcripts in cells of the adult mouse nervous system was assessed by in situ hybridization. Specific hybridization signals were detected in neurons in sensory, sympathetic, and parasympathetic ganglia of the peripheral nervous system. In addition, specific signals were observed in neurons of the ventral and dorsal horns of the spinal cord and of the brain stem, cerebellum, cerebral cortex, hippocampus, dentate gyrus, and olfactory bulb. These results show that the nectin-1 gene is widely transcribed in neurons in adult mouse. Nectin-1 is the only known receptor capable of mediating the entry of all three viruses, HSV-1, HSV-2, and PRV. Its pattern of expression in the nervous system suggests a key role in neurological disease caused by these viruses.

Portable sulphotransferase domain determines sequence specificity of heparan sulphate 3-O-sulphotransferases.

3-O-Sulphates are the rarest substituent of heparan sulphate and are therefore ideally suited to the selective regulation of biological activities. Individual isoforms of heparan sulphate D-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence-specific action, which creates heparan sulphate structures with distinct biological functions. For example, 3-OST-1 preferentially generates binding sites for anti-thrombin, whereas 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes comprise a presumptive sulphotransferase domain and a divergent N-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNA species. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3(A) to generate N1-ST3(A). Similarly, the N-terminal region of 3-OST-3(A) was fused to the sulphotransferase domain of 3-OST-1 to generate N3(A)-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of binding sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming anti-thrombin-binding sites than 3-OST-3(A), indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. N3(A)-ST1 was as active as 3-OST-1, whereas the activity of N1-ST3(A) was as low as that of 3-OST-3(A). Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3(A) and N1-ST3(A) generated gD-binding sites and conveyed susceptibility to infection by HSV-1. Thus sequence-specific properties of 3-OSTs are defined by a self-contained sulphotransferase domain and are not directly influenced by the divergent N-terminal region.

A first step toward understanding membrane fusion induced by herpes simplex virus.

In this issue of Molecular Cell, Carfí et al. present the X-ray structure of the N-terminal domains of HveA, a TNF receptor family member, in complex with herpes simplex virus gD, providing a first step to understanding the herpesvirus mode of membrane fusion.

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.

Visual response properties of neurons in the LGN of normally reared and visually deprived macaque monkeys.

It is now well appreciated that parallel retino-geniculo-cortical pathways exist in the monkey as in the cat, the species in which parallel visual pathways were first and most thoroughly documented. What remains unclear is precisely how many separate pathways pass through the parvo- and magnocellular divisions of the macaque lateral geniculate nucleus (LGN), what relationships-homologous or otherwise-these pathways have to the cat's X, Y, and W pathways, and whether these are affected by visual deprivation. To address these issues of classification and trans-species comparison, we used achromatic stimuli to obtain an extensive set of quantitative measurements of receptive field properties in the parvo- and magnocellular laminae of the LGN of nine macaque monkeys: four normally reared and five monocularly deprived of vision by lid suture near the time of birth. In agreement with previous studies, we find that on average magnocellular neurons differ from parvocellular neurons by having shorter response latencies to optic chiasm stimulation, greater sensitivity to luminance contrast, and better temporal resolution. Magnocellular laminae are also distinguished by containing neurons that summate luminance over their receptive fields nonlinearly (Y cells) and whose temporal response phases decrease with increasing stimulus contrast (indicative of a contrast gain control mechanism). We found little evidence for major differences between magno- and parvocellular neurons on the basis of most spatial parameters except that at any eccentricity, the neurons with the smallest receptive field centers tended to be parvocellular. All parameters were distributed unimodally and continuously through the parvo- and magnocellular populations, giving no indications of subpopulations within each division. Monocular deprivation led to clear anatomical effects: cells in deprived-eye laminae were pale and shrunken compared with those in nondeprived eye laminae, and Cat-301 immunoreactivity in deprived laminae was essentially uniformly abolished. However, deprivation had only subtle effects on the response properties of LGN neurons. Neurons driven by the deprived eye in both magno- and parvocellular laminae had lower nonlinearity indices (i.e., summed signals across their receptive fields more linearly) and were somewhat less responsive. In magnocellular laminae driven by the deprived eye, neuronal response latencies to stimulation of the optic chiasm were slightly shorter than those in the nondeprived laminae, and receptive field surrounds were a bit stronger. No other response parameters were affected by deprivation, and there was no evidence for loss of a specific cell class as in the cat.

Organization of the rat Tage4 gene and herpesvirus entry activity of the encoded protein.

The Tage4 gene (Tumor-Associated Glycoprotein E4) is a member of the immunoglobulin superfamily overexpressed in rat colon tumors and Min mouse intestinal adenomas. The Tage4 cDNA presents approximately 60% identity with the human CD155, a member of the immunoglobulin superfamily coding for a transmembrane protein capable of serving as an entry receptor for poliovirus, porcine pseudorabies virus and bovine herpesvirus 1. We determined the structure of the Tage4 gene. This gene covers approximately 15 kb and is composed of eight exons and seven introns. We also isolated approximately 2 kb of the 5' flanking region of the Tage4 gene and demonstrated the existence of closely clustered transcription start sites. No splicing variant was identified by RT-PCR indicating that the Tage4 gene is transcribed as a unique mRNA. Finally, the protein encoded by the Tage4 gene was tested for ability to mediate entry of several viruses. These structural and functional features of the rat Tage4 gene were compared to those of the human CD155 gene. The results indicated that the Tage4 gene is probably orthologous to the gene for CD155.

Contributions of gD receptors and glycosaminoglycan sulfation to cell fusion mediated by herpes simplex virus 1.

Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.

Cell fusion induced by herpes simplex virus glycoproteins gB, gD, and gH-gL requires a gD receptor but not necessarily heparan sulfate.

To characterize cellular factors required for herpes simplex virus type 1 (HSV-1)-induced cell fusion, we used an efficient and quantitative assay relying on expression of HSV-1 glycoproteins in transfected cells. We showed the following: (1) Cell fusion depended not only on expression of four viral glycoproteins (gB, gD, and gH-gL), as previously shown, but also on expression of cell surface entry receptors specific for gD. (2) Cell fusion required expression of all four glycoproteins in the same cell. (3) Heparan sulfate was not required for cell fusion. (4) Coexpression of receptor with the four glycoproteins in the same cell reduced fusion activity, indicating that interaction of gD and receptor can limit polykaryocyte formation. Overall, the viral and cellular determinants of HSV-1-induced cell fusion are similar to those for viral entry, except that HSV-1 entry is significantly enhanced by binding of virus to cell surface heparan sulfate.

Evaluation of poly(styrene-4-sulfonate) as a preventive agent for conception and sexually transmitted diseases.

A commercial preparation of a sodium polystyrene sulfonate (designated as N-PSS; its molecular weight is 500000 daltons) was tested as an inhibitor of sperm function and as a preventive agent for conception and the transmission of sexually transmitted diseases. The polymer is an irreversible inhibitor of hyaluronidase and acrosin; its IC50 values are 5.7 microg/mL and 0.5 microg/mL, for hyaluronidase and acrosin, respectively. N-PSS is also a stimulus of human sperm acrosomal loss. It produces maximal acrosomal loss at 2.5 microg/mL. Contraception in rabbits is nearly complete when rabbit spermatozoa are pretreated with 0.5 mg/mL of N-PSS before artificial insemination; however, N-PSS does not immobilize spermatozoa at concentrations as high as 50 mg/mL. N-PSS has broad spectrum antiviral and antibacterial activities. Infection by human immunodeficiency virus and herpes simplex virus are inhibited by N-PSS; 3-log reductions are produced by 7 microg/mL and 3 microg/mL, respectively. N-PSS is active against Chlamydia trachomatis and Neisseria gonorrhoeae. At 1 mg/mL, N-PSS inhibits chlamydial infectivity by more than 90%. N-PSS produces a 3-log reduction in gonococcal growth at 15 microg/mL. In contrast, N-PSS (5 mg/mL) does not affect the growth of Lactobacillus (normal component of the vaginal flora). N-PSS can be classified as a noncytotoxic contraceptive antimicrobial agent. These properties justify bringing a polystyrene sulfonate into clinical trials for its evaluation as a preventive agent for conception and several sexually transmitted diseases.

Striking similarity of murine nectin-1alpha to human nectin-1alpha (HveC) in sequence and activity as a glycoprotein D receptor for alphaherpesvirus entry.

A cDNA encoding the murine homolog of human nectin-1alpha (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1alpha, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1alpha but not to control cells, and (iii) cells expressing both murine nectin-1alpha and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.