Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].     

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.     

Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Regions of the CD4 molecule not involved in virus binding or syncytia formation are required for HIV-1 infection of lymphocytes.

Cell surface-expressed CD4 binds to the envelope glycoprotein of HIV-1 and mediates syncytia formation through interacting with membrane expressed HIV-1 gp120. Further possible roles of the CD4 molecule in the process of cell infection by HIV-1 remain poorly understood. In our study we describe two mAb that recognize the V3/V4 domain of the CD4 molecule. Although these mAb do not inhibit gp120-CD4 binding or HIV-1-induced syncytia formation, they inhibit HIV-1 infection of human PBL. These findings suggest that discrete, definable domains of the CD4 molecule may be involved in interactions after HIV-1 envelope binding that lead to virus entry into the cell.     

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.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.     

Heterogeneity of high affinity uptake of L-glutamate and L-aspartate in the mammalian central nervous system.

Characteristics of high affinity uptake of L-glutamate are examined in order to evaluate the possible use of the uptake of [3H]L-glutamate, [3H]L-aspartate or any other suitable [3H]-labelled substrate as a marker for glutamatergic and aspartergic synapses in autoradiographic studies in the mammalian brain. Review of data on substrate specificity indicates the presence of at least two high affinity uptake systems specific for acidic amino acids in the central nervous tissue; one which takes up L-glutamate and L-aspartate and the other which is selective for L-glutamate only. Studies on ionic requirements, too, point to the existence of at least two distinct uptake systems with high affinity for L-glutamate. The Na(+)-dependent uptake system(s) handle(s) both L-glutamate and L-aspartate whereas the Na(+)-independent uptake system(s) show(s) selectivity for L-glutamate only. Available data do not favour the Na(+)-dependent binding of [3H]D-aspartate to thaw-mounted sections of frozen brain tissue as a suitable marker for glutamatergic/aspartergic synaptic nerve endings. However, there are reasons--such as the results of lesion studies and the existence of uptake sites which have a higher affinity for L-aspartate than for D-aspartate--to suggest that Na(+)-dependent binding of [3H]L-aspartate, rather than that of [3H]D-aspartate, should be further investigated as a possible marker for the glutamatergic/aspartergic synapses in the autoradiographic studies using sections of frozen brain.