Induction of telomerase activity in stimulated human lymphocytes precedes expression of topoisomerase II alpha

Telomerase activity has been demonstrated in human immortal cell lines and in tumors, whereas it is generally absent from normal tissues, with the exception of germ cells. Low levels have also been detected in blood and skin cells. In this report we describe up-regulation of telomerase activity in normal human blood lymphocytes by mitogen stimulation. After 24 h of mitogen treatment a strong induction was detectable using the PCR-based telomeric repeat amplification protocol. The level of activity remained almost constant when the cultivation lasted 72 h. By contrast, topoisomerase II alpha was induced later with a maximum expression after 48-72 h. Our data show that telomerase can be induced in normal peripheral lymphocytes prior to expression of the S-phase typical protein topoisomerase II alpha indicating that telomere elongation might be initiated before DNA replication.     

Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element

Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.     

E1A-induced immortalization of rat type II alveolar epithelial cells

Using a retroviral vector expressing the adenoviral 12S E1A gene product the authors have immortalized rat type II alveolar epithelial cells. For a period of time, the immortalized cells retain many of the ultrastructural characteristics of type II cells in situ, including the presence of lamellar bodies. By 250 days in culture, however, neither lamellar bodies, SP-A, nor a phospholipid profile characteristic of surfactant were present. The cell bind the lectin Maclura pomifera and stably express cytokeratins and the E1A gene product. The cell line also has a diploid karyotype, exhibits contact inhibition of growth, and does not grow in soft agar. E1A-immortalized cell lines should prove useful as models for study of certain aspects of type II alveolar epithelial cell function.     

Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase

The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32.     

RNA-binding activity of the E1B 55-kilodalton protein from human adenovirus type 5

The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs. This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.     

Bcl-2 prevents topoisomerase II inhibitor GL331-induced apoptosis is mediated by down-regulation of poly(ADP-ribose)polymerase activity

Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme responsible for DNA strand breaks, has been recently suggested to be crucial for apoptosis induced by a number chemotherapeutic drugs. In this study, we demonstrated that the PARP activity could be evidently elevated with a peak at 6 h when HL-60 cells were treated with a new anticancer drug GL331. Coincident with the peak of PARP activity, an apparent DNA fragmentation and apoptotic morphology were observed in cells treated with GL331. The subsequent apoptotic DNA fragmentation induced by GL331 could be completely blocked by transfecting cells with anti-sense PARP retroviral vector or by treating cells with PARP inhibitor, 3-aminobenzamide (3-AB). This blocking effect thus suggests that activation of PARP was critically involved in GL331-induced apoptosis. The fact that Bcl-2 has been found to antagonize cell death induced by a wide variety of agents, accounts for why we examined whether if Bcl-2 could antagonize GL331 effects. Interestingly, ectopic overexpression of Bcl-2 in either HL-60 or U937 cells caused in resistance towards GL331-elicited DNA fragmentation and cytotoxic effect. Additionally, Bcl-2 also attenuated the poly(ADP-ribosyl)ation of PARP itself as well as Histone H1 at the early period of drug treatment. However, Bcl-2 did not influence the extent of DNA strand breaks induced by GL331 in either control or Bcl-2-overexpressing cells. In addition, analysis of basal PARP activity in control and several Bcl-2 overexpressing clones revealed that Bcl-2 down-regulated PARP activity under the condition without DNA damages. Above findings suggest that poly(ADP-ribosyl)ation of nuclear targets is important for apoptosis induced by DNA-reactive anticancer drugs.     

Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.     

Rapid isolation of genomic clones for individual members of human multigene families: identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family

We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.     

E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.     

Crystal structure of endo-beta-N-acetylglucosaminidase F1, an alpha/beta-barrel enzyme adapted for a complex substrate

Endo-beta-N-acetylglucosaminidase F1 (Endo F1) is an endoglycosidase, secreted by Flavobacterium meningosepticum, that cleaves asparagine-linked oligosaccharides after the first N-acetylglucosamine residue. The enzyme is selective for high-mannose oligosaccharide chains. The crystal structure of Endo F1 has been determined at 2.0-A resolution. The molecular fold consists of a highly irregular alpha/beta-barrel, a commonly observed motif consisting of a cyclic 8-fold repeat of beta-strand/loop/alpha-helix units with an eight-stranded parallel beta-barrel at the center. Endo F1 lacks two of the alpha-helices, those of units 5 and 6. Instead, the links after beta-strands 5 and 6 consist of a short turn followed by a section in an extended conformation that replaces the helix and a long loop at the bottom of the molecule. The absence of any excursion on top of the molecule following beta-strands 5 and 6 results in a pronounced depression in the rim of the barrel. This depression forms one end of a shallow cleft that runs across the surface of the molecule, over the core of the beta-barrel to the area between the loops of units 1 and 2. The active site residues, Asp130 and Glu132, are located at the carboxyl end of beta-strand 4 and extend into this cleft. These residues are surrounded by several tyrosine residues. The cleft area formed by loops 1 and 2 is lined with polar residues, mainly asparagines. The latter area is thought to be responsible for oligosaccharide binding and recognition while the protein moiety of the substrate would be located outside the molecule but adjacent to the area of loops 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Search for alpha 1-adrenoceptor subtypes selective antagonists: design, synthesis and biological activity of cystazosin, an alpha 1D-adrenoceptor antagonist

Two novel quinazolines (2 and 3) related to both prazosin and its open analogue 1 were synthesized, and their biological profile at alpha 1-adrenoceptor subtypes was assessed by functional assays in rat isolated tissues, namely prostatic vas deferens (alpha 1A), spleen (alpha 1B) and aorta (alpha 1D). Furthermore, the binding profile of 3 was assessed at native alpha 2 and D2 receptors, and cloned human 5-HT1A receptors, in comparison to prazosin, (+)-cyclazosin, 1 and BMY 7383. It turned out that the cystamine-bearing quinazoline 3 (cystazosin) has a reversed affinity profile relative to (+)-cyclazosin owing to a higher affinity for alpha 1D-adrenoceptors and a significantly lower affinity for the alpha 1A and alpha 1B subtypes. Furthermore, in comparison to BMY 7378, cystazosin (3) displays a much better specificity profile since it has lower affinity for D2 and 5-HT1A receptors.     

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.     

Stable restoration of pyruvate dehydrogenase complex in E1-defective human lymphoblastoid cells: evidence that three C-terminal amino acids of E1 alpha are essential for the structural integrity of heterotetrameric E1

In an attempt to restore pyruvate dehydrogenase complex (PDHC), expression vectors carrying wildtype E1 alpha cDNA (pRAWT) or 1162ins-mutant (pRA1162) were introduced into human lymphoblastoid cells which had a 4-bp insertion after nucleotide 1162 (1162ins) of E1 alpha cDNA, 28% of normal PDHC activity, and undetectable levels of both E1 alpha and E1 beta proteins. The amount of E1 alpha mRNA transcribed from the introduced cDNA was approximately 25 times greater than that transcribed from the endogenous gene. The PDHC activity of pRAWT-transformed cells increased to the normal level whereas this activity increased to 55% of the control in pRA1162-transformed cells. Mitochondria from pRAWT-transformed cells contained normal amounts of both the E1 alpha and the E1 beta subunits. These results suggest that the three C-terminal amino acids of E1 alpha, which were absent from 1162ins-mutant protein, may be important for the structural integrity of E1 and that a large amount of normal subunit, compared to the endogenous mutant enzyme, must be expressed to restore a multienzyme complex.     

Effects of prostaglandins E1, E2, F2 alpha and arachidonic acid on aryl hydrocarbon hydroxylase activity of intestinal and hepatic microsomes from male rats

The in vitro effects of prostaglandins E1, E2, F2 alpha and arachidonic acid on microsomal aryl hydrocarbon hydroxylase (AHH) activity from rat liver and intestinal mucosa were examined. PGE1 and PGE2 stimulated AHH activity of intestinal microsomes, while both prostaglandins had an inhibitory effect on AHH activity of liver microsomes. PGF2 alpha had little effect on AHH activity of intestinal and hepatic microsomes. Arachidonic acid exerted strongly inhibitory effects on AHH activity of both intestine and liver, with the greatest inhibition being exerted against intestinal AHH. Kinetic studies demonstrated that the inhibition of hepatic AHH activity by PGE1 at concentrations of 0.10 mM or less was non-competitive.     

Inhibition of CYP2E1 activity does not abolish pulsatile urine alcohol concentrations during chronic alcohol infusions

Continuous, long-term intragastric infusions of ethanol leads to a two-step induction of hepatic cytochrome P450 2E1 (CYP2E1) that is correlated with blood ethanol concentrations (BECs) and urine alcohol concentrations (UECs). In addition, long-term and continuous ethanol infusion does not produce a steady-state BEC, but results in pulsatile BECs and UECs having peak-peak duration of approximately 6 days and ethanol concentrations ranging from near zero to over 500 mg/dl. In the present study, rats were treated with ethanol (levels reaching 13 g.kg-1.day-1) for 38 days in the presence of compounds reported to block CYP2E1 activity or expression, to study the possible involvement of CYP2E1 in the pulsatile BECs. The inhibitors used were chlormethiazole (CMZ); diallysulfide (DAS); phenethyl isothiocyanate (PET) and dihydrocapsacin (DHC). Hepatic microsomal metabolism of carbon tetrachloride and p-nitrophenol, as well as mean CYP2E1 apoprotein levels, were significantly greater (P < or = 0.05) in ethanol-treated rats than in control rats, whereas rats treated with DAS, CMZ or PET had significantly (P < or = 0.05) reduced p-nitrophenol and carbon tetrachloride metabolism and lower CYP2E1 apoprotein levels compared to those of ethanol controls. UECs were variable in all ethanol-treated groups and there was a typical pulsatile pattern that had a mean interpulse interval (the number of days between the peaks of two consecutive pulses) ranging over 5.4 +/- 0.3-6.0 +/- 0.7 days and a mean amplitude (nadir to peak UEC) of 415 +/- 39-337 +/- 33 mg/dl. None of the putative CYP2E1 blockers altered the pulsatile nature of ethanol in urine. Our results suggest that pulsatile UECs are not the result of variations in the amount of CYP2E1.