Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation.

Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte-macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.     

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl     

Protein synthesis modulates the biological effectiveness of the combined action of hyperthermia and high-LET radiation.

The combined effects of heavy-ion radiation and hyperthermia on the survival of CHO-SC1 cells and its temperature-sensitive (ts) mutant tsH1 cells were studied using accelerated neon ions followed by mild heating at 41.5 degrees C. The sequence of application of heat and high-LET radiation is significant to cell-killing effects. Heat applied to cells prior to irradiation with neon plateau ions (LET = 32 keV/microns) was less effective than heat applied immediately after irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal sensitization. When protein synthesis was shut down in tsH1 cells, the thermal enhancement of cell killing by high-LET radiation was the same regardless of the sequence. The thermal enhancement of radiation-induced cell killing was LET-dependent for the SC1 cells, but this was not clearly demonstrated in the tsH1 cells. Furthermore, the RBE of heated SC1 cells varied with LET and reached a maximum of greater than 3 at 80 keV/microns. In the absence of protein synthesis, the maximum RBE value was reduced to 2.6. These results suggest that the accumulation of cellular damage caused by exposure to densely ionizing particles with increasing LETs can be potentiated with active protein synthesis during postirradiation heat treatment.     

Identification of nitroxide radioprotectors.

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.     

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.     

Methylprednisolone restores sensitivity to beta-adrenergic agonists in Basenji-Greyhound dogs.

This study investigated the effect of chronic methylprednisolone treatment on the ability of albuterol and aminophylline to inhibit methacholine-induced airway constriction in Basenji-Greyhound (BG) dogs in vivo. Pulmonary responsiveness to methacholine was measured in five untreated BG dogs and in the same dogs pretreated with albuterol or aminophylline (which has been shown in this model to release endogenous catecholamines). Each dog was studied before, during, and after daily subcutaneous methylprednisolone for 6 wk. Changes in pulmonary resistance and dynamic compliance with methacholine aerosol challenge were measured. Neither baseline pulmonary function nor pulmonary responsiveness to aerosolized methacholine was significantly altered by albuterol, aminophylline, or chronic methylprednisolone administration alone. However, pretreatment with albuterol or aminophylline significantly attenuated airway responses to methacholine in BG dogs chronically receiving methylprednisolone. Because the reduced sensitivity to albuterol and aminophylline was restored by chronic methylprednisolone treatment, we conclude that at least part of the beneficial effects of corticosteroids on airways in BG dogs is through modulation of beta-adrenergic function.     

Multiple hormone actions: the rises in cAMP and Ca++ in MDCK-cells treated with glucagon and prostaglandin E1 are independent processes.

Glucagon and prostaglandin E1 stimulate adenylate cyclase in Madin-Darby canine kidney cells with an approximate EC50 of 3*10(-8) and 1*10(-7) M respectively. The rise in cAMP is accompanied by a transient rise in intracellular Ca++ measured with the fluorescent calcium indicator Indo-1. A comparable increase in intracellular Ca2+ without a rise in cAMP occurs with the cholinergic agonist carbamylcholine. Stimulation of adenylate cyclase by the beta-adrenergic agonist isoproterenol or directly by forskolin has no effect on intracellular Ca++. By all criteria studied the rise in intracellular Ca++ and the increase in cAMP are independent from each other.