The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor.

All five functional domains of the low density lipoprotein (LDL) receptor were assembled in their modern form more than 350 million years ago, as revealed from the sequence of two cloned cDNAs from the frog Xenopus laevis. The two cDNAs appear to represent duplicated copies of the LDL receptor gene that arose when the entire genome of Xenopus duplicated approximately 30 million years ago. Both frog LDL receptors bound Xenopus LDL with high affinity and human LDL with lower affinity when expressed in monkey COS cells. The receptors also showed high affinity for rabbit beta-migrating very low density lipoprotein and canine apoE-HDLc, both of which contain apolipoprotein E. Each of the seven cysteine-rich repeats in the ligand binding domain of the Xenopus receptors resembles its counterpart in the human, indicating that these repeats had already acquired their independent structures by the time of amphibian development. The cytoplasmic tail of both Xenopus receptors is 86% identical to the human, including the FDNPVY sequence necessary for internalization in coated pits. The attainment of a fully developed receptor structure in Xenopus suggests that earlier forms of the receptor may exist in animals that are older than amphibians. An accompanying paper demonstrates that expression of both Xenopus receptor genes is controlled by a sterol regulatory element that closely resembles the human sequence (Mehta, K.D., Brown, M.S., Bilheimer, D.W., and Goldstein, J.L. (1991) J. Biol. Chem. 266, 10415-10419).     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Immunodeficiency in the absence of high viral load in pig-tailed macaques infected with Simian immunodeficiency virus SIVsun or SIVlhoest

Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4(+) lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as Mycobacterium avium or Pneumocystis carinii infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.     

Murine [corrected] myeloid dendritic cell-dependent toll-like receptor immunity is preserved with aging

The immune response is the result of the interplay between innate and adaptive immunity, yet the impact of aging on this interaction is unclear. Addressing this fundamental question will be critical for the development of effective vaccines for the rapidly rising older subpopulation that manifests increased prevalence of malignancies and infections. Therefore, we undertook the current study to investigate whether aging impairs toll-like receptor (TLR) function in myeloid dendritic cells and whether this leads to reduced T-cell priming. Our results demonstrate that innate TLR immune priming function of myeloid bone marrow derived and splenic dendritic cells (DC) is preserved with aging using both allogeneic and infectious murine experimental systems. In contrast, aging impairs in vitro and in vivo intrinsic T-cell function. Therefore, our results demonstrate that myeloid DCs manifest preserved TLR-mediated immune responses with aging. However, aging critically impairs intrinsic adaptive T-cell function.     

Proton block and voltage gating are potassium-dependent in the cardiac leak channel Kcnk3

Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (tau = approximately 5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels.     

Intracisternal administration of cholecystokinin-8 counteracts the central cardiovascular effects of adrenaline and NPY. A study based on the coexistence of cholecystokinin, phenylethanolamine N-methyltransferase and neuropeptide Y immunoreactivity in neurons of the nucleus tractus solitarius

(1) In the present study the occlusion method was employed to evaluate the overall coexistence of neuropeptide Y and phenylethanolamine-N-methyl transferase, neuropeptide Y and tyrosine hydroxylase as well as cholecystokinin and phenylethanolamine-N-methyl transferase immunoreactivity in nerve cell bodies of the dorsal subnuclei of the nucleus tractus solitarius of the male rat. A high degree of coexistence was established for neuropeptide Y/phenylethanolamine-N-methyl transferase, cholecystokinin/phenylethanolamine-N-methyl transferase and for tyrosine hydroxylase/neuropeptide Y immunoreactivity. (2) Sulfated [¹²I]cholecystokinin-8 was used as radioligand to study the densities of cholecystokinin-8 binding sites in the dorsal medulla oblongata by means of quantitative receptor autoradiography. High densities of binding sites were observed in parts of the nucleus tractus solitarius and in the area postrema. Labeling was also observed in the dorsal motor nucleus of the vagus. (3) In the physiological studies adrenaline (0.15–1.0 nmol), neuropeptide Y (0.075–0.75 nmol) and sulfated cholecystokinin-8 (0.3–3.0 nmol) were administered alone or in combination with neuropeptide Y or adrenaline intracisternally into -chloralose anaesthetized male rats. Especially the hypotensive and bradycardic responses of adrenaline were counteracted in the adrenaline/cholecystokinin co-treated animals, whereas the cardiovascular effects of neuropeptide Y when co-administered with cholecystokinin-8 (0.3 nmol) appeared to be more resistant to the antagonistic effect of cholecystokinin 8. In addition, cholecystokinin-8 further enhanced the neuropeptide Y-induced bradynpnea and increase in the tidal volume.

The present results indicate the existence of neuropeptide Y, adrenaline and cholecystokinin-8 immunoreactivity in the same neurons of the dorsal subnuclei of the nucleus tractus solitarius. Furthermore, binding sites for cholecystokinin-8 seem to at least partly co-distribute with -2 adrenergic and neuropeptide Y binding sites in the nucleus tractus solitarius. In the functional analysis, an antagonistic interaction between cholecystokinin-8 and adrenaline as well as between cholecystokinin and neuropeptide Y is demonstrated opening up the possibility that cholecystokinin peptides act as intrinsic modulators in the putative cholecystokinin/neuropeptide Y/adrenaline synapses in the nucleus tractus solitarius.     

Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.