Human telomeres maintain their overhang length at senescence.

Normal human cells in culture enter replicative senescence after a finite number of population doublings. The exact molecular mechanisms triggering the growth arrest are poorly understood. A recent report on the disappearance of the G-rich 3' telomeric overhang in senescent cells led to the hypothesis that loss of the 3' G-rich overhang is the molecular signal that triggers senescence. Here, we describe a quantitative assay to measure the length of the G-rich 3' telomeric overhangs from cultured cells. Using both this assay and the conventional nondenaturing hybridization assay for measuring G-rich overhangs, we show that normal human fibroblasts can maintain their overhangs at senescence. Furthermore, cells do not lose their overhangs when they bypass senescence after the inactivation of p53 and Rb. We thus conclude that a global reduction in overhang length is not the molecular signal that triggers replicative senescence.

Nuclear transport of single molecules: dwell times at the nuclear pore complex.

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 +/- 0.2 and 7.1 +/- 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM).

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

Studies on mimicry of naturally occurring annonaceous acetogenins: non-THF analogues leading to remarkable selective cytotoxicity against human tumor cells.

A class of structurally simplified analogues of the naturally occurring annonaceous acetogenins were developed, amongst which some non-THF analogues showed remarkable cytotoxicities against tumor cell lines, as well as good selectivity between human tumor cells and normal cells. The synthetic routes were significantly shortened because of the removal of the chiral centers bearing the THF rings on the natural templates. This simplification also provides access to the parallel synthesis of these mimics by a combinatorial strategy. The remaining stereogenic centers at the positions alpha to the ethereal links were introduced by the Chiron approach from the easily accessible chiral building blocks 6a and/or 6b, made in turn from L-ascorbic acid or D-mannitol, while the one in the butenolide segment was taken from L-lactate. All four diastereomeric non-THF analogues 2a-2d showed remarkable activity against the HCT-8 cell line, and better differentiation was found when testing against the HT-29 cell line. It was also discovered that both the butenolide and ethylene glycol subunits play essential roles in the cytotoxicities against tumor cell lines, while the 10-substituted hydroxy group and the absolute configuration of methyl group at the butenolide moiety are less important for their activity.

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.

Immunomodulatory agents for prophylaxis and therapy of infections.

The advent of the antibiotic era ushered in a shift towards non-pathogen-specific therapy of infectious diseases. This led to an overt emphasis on targeting microbial pathogens while strategies directed towards enhancing host immunity were neglected. In an effort to decrease sole reliance on antimicrobials, the time has come for a critical reappraisal of nonantibiotic, albeit immune response-enhancing substances. The diverse array of natural, synthetic, and recombinant immunomodulators discussed in this review succinctly demonstrate the potential of these agents to stimulate host defense mechanisms for prophylaxis and treatment of various microbial infections.

Molecular cloning, sequence analysis, expression, and tissue distribution of suppressin, a novel suppressor of cell cycle entry.

Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990) J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5'- and 3'-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at approximately 2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator in Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types.

Functional roles of phenylalanine(7) of thymic humoral factor-gamma 2 in the impaired blastogenic response of uremic T-lymphocytes.

The peptide analogs of thymic humoral factor-gamma 2 (THF-gamma 2) in which phenylalanine residue at the 7th position are replaced by phenylglycine (Phg), homophenylalanine (Hph), and 1-naphthylalanine (1-Nal) were synthesized by a solid-phase method and the immunological significance of the aromatic amino acid of this position was comparatively investigated. The in vitro restoring effect of the synthetic peptides on the impaired phytohemagglutinin (PHA) response of T-lymphocytes from uremic patients was tested. The observed activities of these peptides were in order (1-Nal7) thymic humoral factor [THF]-gamma 2 > 4-Fluoro (Phe7) THF-gamma 2 > THF-gamma 2. However, the other two analogs, [Phg7] THF-gamma 2 and [Hph7] THF-gamma 2, had no restoring effect even at a higher concentration.

Syntheses and immunological effects of thymic humoral factor-gamma 2 and its Phe7-substituted analogues.

Thymic humoral factor-gamma 2 and five analogues modified at position 7 with various phenylalanine derivatives were synthesized by a solid-phase method. The synthetic peptides were tested for their effects on the impaired blastogenic response of phytohemagglutinin-stimulated T lymphocytes of uremic patients with infectious diseases. The synthetic thymic humoral factor-gamma 2 enhanced the blastogenic response of T lymphocytes in the blood of the 2 patients tested. Of the synthetic peptides, [Phe(4F)7]thymic humoral factor-gamma 2 exhibited the most potent effect.

[Role of the non-peptide thymic factor--hypoxanthine system in the proliferation of mature thymocytes of mice and rats]. / Vlyianye khymycheskoi modifykatsyy ostatkov arhinina fibrinohena i eho DH-frahmenta na ksorost' hidroliza étukh belkov plazminom.

The incorporation of [8-14C]hypoxanthine radioactive label into the nucleic acids of the mature thymocytes (that do not bind peanut agglutinin) of CBA mice and Wistar rats and the action of non-peptide thymic mitogenic factor (factor) on this process have been studied. It was shown, that the factor, shifting AMP catabolism towards hypoxanthine accumulation, accelerated by 50% the incorporation of hypoxanthine radioactive label into nucleic acids during 3-hour incubation. It is concluded that the factor being an activator of the hypoxanthine accumulation, abrogates the purine limits in thymocytes and activates the "salvage pathway" of purine synthesis which is the primary pathway in thymocytes. The factor is assumed to act as the mitogen on one of the stages of nucleic acids or nucleotides synthesis, but not to act on the guanine-hypoxanthine phosphoribosyl transferase. It was shown that the intrathymocyte ratio of hypoxanthine and factor concentrations (hypoxanthine/factor) is higher in rats than in mice. The dependence of proliferative activity on relative levels of both substances was revealed. The stimulation of cell proliferation by the system: factor--hypoxanthine is one of the mechanisms of thymus regeneration at the expense of mature thymocyte population.

Histones and related preparations interfere with immunoassays for peptide hormones.

We report here that histones and certain related preparations generate a consistent interference with radioimmuno (RIA), immunoradiometric (IRMA), and enzyme-linked immunosorbent (ELISA) assays for a number of peptide hormones. Histones H1, H2A, H2B, H3, HIIA, HIIS, protamine, and the related preparations homeostatic thymus hormone and peptide MB35 generated a dose-dependent signal in both the human corticotropin-releasing hormone (CRH) and the human adrenocorticotropic hormone (ACTH) IRMA. This signal was not affected when the linker antiserum was removed from the IRMA reagent mixture, thus proving that the signal was not due to cross-reaction or sample contamination with CRH or ACTH. The above histone preparations, as well as protamine, but not ubiquitin, also generated a strong negative interference with RIAs for ACTH, CRH, rat growth hormone (rGH), and rat prolactin (rPRL). In an ELISA system for the thymic peptide facteur thymique sérique, histones and protamine again showed a strong interfering activity. When known amounts of rGH, rPRL, and hACTH were dissolved in charcoal-washed horse serum or supernatants from rat liver homogenates (centrifuged 1 h at 10,000 x g), and the corresponding RIAs and IRMA (for ACTH) were performed in the absence or presence of histones HIIA and HIIS (at 1 mg/ml level), an interfering activity of histones was again observed. We conclude that histones and some related peptide preparations have, when present in biologic fluids, a significant capacity to interfere with peptide immunoassays.

[The effect of new synthetic fragments of thymus hormones on the proliferative response of lymphocytes]. / Vliianie novykh sinteticheskikh fragmentov timusnykh gormonov na proliferativnyi otvet limfotsitov.

Influence of three new artificial fragments of beta-thymosines Phe-Asp-Lys-Ala, Glu-Lys-Phe-Asp-Lys and Thr-Leu-Pro-Thr on phytohemagglutinin-induced proliferative response of human lymphocytes was studied. These peptides studied either stimulated or inhibited incorporation of 3H-thymidine in cell cultures of human lymphocytes. Possible mechanisms of these effects of the peptides on lymphocyte proliferation are discussed.

The thymus hypocholesterolemic factor (TphF): a bovine thymic superoxide dismutase active on HMG-CoA reductase.

1. This work describes the further biochemical characterization of a new calf thymus protein (TphF) and its primary structure. 2. The amino acid sequences, obtained after sequence analysis of peptides derived from the endoproteinase Lys-C digestion, were subjected to a "Protein Data Bank Search" and were found to be identical with regions of bovine superoxide-dismutase (SOD). 3. These data together with those showing the identical electrophoretic migration of SOD and TphF, their same isoelectric point and their immunoreactivity with anti-SOD antibodies, confirm the similarity of these two proteins.

Selective photochemical modification by trichloroethanol of tryptophan residues in proteins with a high tyrosine-to-tryptophan ratio.

We present an improved procedure for the selective modification of tryptophan residues in proteins. A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol. This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct. Except for tyrosine, no reaction with other amino acid side chains are known. Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses. Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density. Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved. Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes.

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.

Purification and partial characterization of an avian thymic hormone. Avian thymic hormone.

An avian thymic hormone, originally designated the T1-antigen, was purified from chicken thymus by Sephadex G-75-40 chromatography and affinity chromatography, following enrichment by heat and pH treatments. It was characterized as an acidic polypeptide rich in phenylalanine, alanine and serine, lacking in histidine, tryptophan, methionine and cysteine, and having a blocked N-terminal amino acid. The hormone also was rich in hydrophobic amino acid residues, which gave it a propensity to form aggregates. Its molecular weight was estimated by gel electrophoresis and low speed sedimentation equilibrium to be 12-13 Kd, and by molecular sieving chromatography to be 15-16 Kd. The hormone was lacking in carbohydrates and amino sugars.

The amino acid sequence of avian thymic hormone, a parvalbumin.

The complete amino acid sequence of 'avian thymic hormone' (ATH), a protein from thymus tissue that appears to promote immune maturation in chicken bone marrow cells in culture, is presented. The sequence was obtained from sequences of ATH peptides isolated by HPLC after tryptic, chymotryptic, peptic or S aureus V8 protease digestions. The protein is a parvalbumin consisting of 108 residues with a blocked amino terminus, a single cysteine, tyrosine, proline and arginine and no histidine, methionine or tryptophan. This is the first amino acid sequence of a parvalbumin which is not derived from muscle tissue.