Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: a molecular model for latency

U1 and ACH-2 cells are subclones of HIV-1-infected monocyte/macrophage-like and T lymphocyte cell lines, respectively, which express the HIV-1 genome at very low levels. We have examined whether they might provide a model of HIV-1 latency. The patterns of HIV-1-specific RNA expressed in these cells consisted of singly and multiply spliced RNA species, with little or no full-length genomic RNA. Upon stimulation with agents that activate the HIV-1 long terminal repeat in these cells, a marked rise in the amount of small mRNAs, encoding the viral regulatory proteins, preceded the increase in the unspliced RNA. Thus, U1 and ACH-2 cells maintain HIV-1 in a state equivalent to the early phase of a lytic infection and, after stimulation, recapitulate the events of a single cycle infection of highly susceptible target cells.     

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.     

Transport and steady-state concentration of plasma proteins in the vitreous humor of the chicken embryo: implications for the mechanism of eye growth during early development

The nature and origin of the proteins of the vitreous humor were examined in chickens during embryonic and early posthatching stages. The major proteins of the vitreous humor were similar in electrophoretic mobility to plasma proteins at all ages examined. Earlier studies from our laboratory and experiments described below showed that plasma proteins continuously entered and left the eye throughout its development. From these data it was concluded that the majority of vitreous-humor proteins were derived from the blood. The protein concentration of the vitreous humor was 13% of that of the plasma from embryonic Days 6 through 15 (E6 through E15). After E15, the relative protein concentration in the vitreous humor declined with respect to the plasma and reached 4% of the plasma protein concentration at hatching. Several possibilities were considered to account for how proteins can rapidly enter and leave the eye, yet maintain a steady-state concentration in the vitreous humor that is less than one-seventh of that in the blood.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Antigenic probes locate a serum-gelsolin-interaction site on the C-terminal part of actin.

The implication of part of the C-terminal of actin (within the 285-375 sequence) in the interaction of serum gelsolin was investigated by the use of specific antibodies. These antibodies were directed against two or three discrete epitopes, one of which was specific for skeletal-muscle actin. Some of these epitopes were found to be near the serum gelsolin-actin interface. Thus it can be assumed that part of the C-terminal of actin is exposed at the barbed end of the actin filament. The interaction between tropomyosin and actin was also studied.     

Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilms'' tumor cells.

Children with associated Wilms' tumor, aniridia, genitourinary malformations, and mental retardation (WAGR syndrome) frequently have a cytogenetically visible germ line deletion of chromosomal band 11p13. In accordance with the Knudson hypothesis of two-hit carcinogenesis, the absence of this chromosomal band suggests that loss of both alleles of a gene at 11p13 causes Wilms' tumor. Consistent with this model, chromosomes from sporadically occurring Wilms' tumor cells frequently show loss of allelic heterozygosity at polymorphic 11p15 loci, and therefore it has been assumed that allelic loss extends proximally to include 11p13. We report here that in samples from five sporadic Wilms' tumors, allelic loss occurred distal to the WAGR locus on 11p13. In cells from one tumor, mitotic recombination occurred distal to the gamma-globin gene on 11p15.5. Thus, allelic loss in sporadic Wilms' tumor cells may involve a second locus on 11p.