A highly selective telomerase inhibitor limiting human cancer cell proliferation.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Human telomerase contains two cooperating telomerase RNA molecules.

Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the telomerase reverse transcriptase (TERT) protein subunit. We demonstrate that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules. Significantly, a telomerase heterodimer reconstituted from wild-type and mutant telomerase RNA is barely active when compared with the wild-type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We discuss models that may explain the biological and enzymatic roles of telomerase dimerization.

Stability of employment after brain injury: a 7-year follow-up study.

Forty-three patients with severe traumatic brain injury (n = 24), cerebrovascular diseases (n = 15), or other acquired brain damage (n = 4) were followed-up 7-8 years after neuropsychological rehabilitation including a vocational re-entry programme. Current vocational status and work history since rehabilitation were investigated by means of a structured interview. Before interview, the patients were classified on the basis of medical records into four groups: (A) patients with minor residual neuropsychological impairments, (B) patients with minor impairments but psychopathological symptoms, (C) patients with persistent neuropsychological impairments showing no psychopathological symptoms, and (D) patients with persistent impairments and psychopathological symptoms. For patients in group A, a good, and for those in group D, a poor long-term employment outcome was predicted, while no predictions were made for the intermediate groups. Of the 43 interviewed subjects, 16 (37%) reported a stable return to work at pre-morbid level and seven (16%) at a lower level. In eight patients (19%), persisting difficulties in maintaining work were documented. Twelve subjects (28%) had retired within a period of 2 years after work trial. The relationship between patient classification and long-term employment outcome was only weak. Four out of 11 patients with a good prognosis (group A) experienced vocational adjustment problems or had retired. Three out of 10 patients with a poor prognosis (group D) were able to continue successfully with their previous jobs. These cases are described in detail. The employment outcome of the intermediate groups was very heterogeneous. The results suggest that particular attention should be paid to the long-term consequences of a reduced capacity for work, even if minor in degree. The success of patients despite a poor prognosis illustrate unsolved problems in relation to the ecological validity of neuropsychological measures of executive dysfunctions.

A perinatal education consortium: improved resource utilization.

Maternal Newborn Nurse Professionals of Southeastern Michigan is an organization of nurses from more than 30 hospitals. The organization formed a committee to provide a comprehensive, cost-effective educational program for new nurses or nurses who were being cross trained, which would improve resource utilization in the region. Twice a year the organization offers a 5-day educational program covering antepartum, fetal monitoring, intrapartum, postpartum, and neonatal issues. The development, implementation, and evaluation of the program are outlined.

Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.

Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit.

EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.

Engineering of variants of the restriction endonuclease EcoRV that depend in their cleavage activity on the flexibility of sequences flanking the recognition site.

The present work describes mutants of the restriction enzyme EcoRV that discriminate very efficiently between oligodeoxynucleotide substrates with an EcoRV recognition sequence in different sequence context. All of these EcoRV variants harbor substitutions at position 226, where in the cocrystal structure of the specific EcoRV/DNA complex an arginine contacts the backbone of the DNA substrate upstream of the recognition sequence, and cleave an oligodeoxynucleotide with an EcoRV site in a nonflexible sequence context (the recognition site being flanked by runs of A and T) with much higher catalytic efficiency (kcat/Km) than an oligodeoxynucleotide with an EcoRV site in a flexible sequence context (the recognition site being flanked by runs of AT), in contrast to the wild-type enzyme, that cleaves both substrates with the same catalytic efficiency. Steady-state and single-turnover kinetics indicate that the enhanced selectivity of the mutants is due to the catalytic step of the reaction. It is possible to enhance the discriminatory power of these EcoRV variants through the choice of appropriate reaction conditions, in particular low salt concentration and low reaction temperatures. It must be emphasized that the enhanced selectivity of these EcoRV variants toward EcoRV sites in a flexible and nonflexible sequence context, respectively, is not only seen with oligodeoxynucleotides, but also with plasmid substrates.

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides.

Synthetic oligodeoxynucleotides with single methyl phosphonate (mp) substitutions were used for an analysis of the contribution of phosphate contacts to the recognition of the cleavage site by the restriction endonuclease EcoRV. Only in the last position within the recognition sequence, is the methyl phosphonate substitution tolerated by the enzyme. The wild-type enzyme cleaves the Sp diastereomer of the oligodeoxynucleotide GACGATATmpCGTC and the unmodified sequence with equal rates, whereas the Rp diastereomer is cleaved much more slowly. Inspection of the crystal structure of an EcoRV-DNA complex revealed that the non-bridging oxygen atoms of the phosphodiester bond between the T and C bases are in hydrogen bonding distance of the hydroxyl group of the amino acid Thr94. We therefore tried to engineer a variant of EcoRV that would prefer a methyl phosphonate linkage over a normal phosphodiester bond and produced mutants with amino acid exchanges at position 94. One of them, Thr94Val, shows a dramatically reduced activity towards the unmodified DNA and does not accept the Rp diastereomer, but cleaves the Sp diastereomer with the same rate as wild-type EcoRV. Its selectivity, i.e. the ratio of cleavage rates determined for the unmodified and modified substrates, differs by three orders of magnitude from that of the wild-type enzyme.

Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.

Engineering novel restriction endonucleases: principles and applications.

Restriction endonucleases cleave DNA with remarkable sequence specificity. In this review, we summarize the status of, and prospects for, engineering restriction endonucleases with new specificities. Such variants could be of considerable commercial value because restriction enzymes are among the most frequently used enzymes in molecular biology, and not all the desirable specificities are available. While it has not yet been possible to effect specificity changes, mutant have been described that (1) exhibit relaxed specificity, (2) favour modified substrates over their natural substrates, (3) discriminate between cleavage sites located in different sequences, (4) prefer metal ions other than Mg2+ as cofactors for cleavage, or (5) possess site-specific DNA-nicking activity.

Probing the indirect readout of the restriction enzyme EcoRV. Mutational analysis of contacts to the DNA backbone.

According to the crystal structure of the specific EcoRV.DNA complex, not only the functional groups of the nucleobases but also the phosphate groups of the DNA backbone are contacted by the enzyme. To examine the contribution of backbone contacts to substrate recognition and catalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resulting EcoRV mutants and characterized them with respect to their DNA binding and cleavage activity. According to our steady state kinetic analysis, there are strong interactions between three basic amino acid residues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV.DNA complex. These contacts are important in both the ground state and the transition state. Other, uncharged residues (Thr-93 and Ser-112), which could be involved in hydrogen bonds to the phosphate groups, are needed primarily to stabilize the transition state. An especially important amino acid residue is Thr-37, which seems to couple recognition to catalysis by indirect readout.

DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme.

In contrast to many other type II restriction endonucleases, EcoRV binds specifically to DNA only in the presence Mg2+. According to the co-crystal structure of an EcoRV-DNA complex, Mg2+ ion(s) bind to the active site of EcoRV liganded by Glu45, Asp74, and Asp90. Here we present experimental evidence suggesting that the EcoRV-DNA complex also interacts with Mg2+ ions at other sites: (i) We have prepared an EcoRV triple mutant, in which all acidic amino acids in the catalytic center are replaced by alanine. This mutant is catalytically inactive. It binds nonspecifically to DNA in the absence of Mg2+, whereas it binds specifically to DNA in the presence of Mg2+. This means that Mg2+ induces specific DNA binding in this mutant, although all Mg2+ ligands in the catalytic center are removed. Therefore, additional interactions between Mg2+ and the EcoRV-DNA complex probably occur at sites distinct from the catalytic center. (ii) We have measured the specific and nonspecific DNA binding constants of EcoRV and of the triple mutant in the presence and absence of Mg2+. Mg2+ reduces nonspecific binding by 3-4 orders of magnitude, presumably because Mg2+ ions bound to the DNA have to be released upon complex formation. In contrast, the specific binding of the wild-type enzyme and the triple mutant is increased in the presence of Mg2+. This result can only be explained if a Mg2+ ion binds to the specific EcoRV-DNA complex probably at a site distinct from the catalytic center.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates.

According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.

[Clinical case report: unilateral malformation of the eye of a thoroughbred foal]. / Klinischer Fallbericht: Einseitige Missbildung am Auge eines Vollblutfohlens.

A unilateral malformation of the eye of a thoroughbred foal is described. The specific form of the tiny lens we named, "lenticulus". It is correlated with a maximal unchangeable mydriasis. The bulb shows physiological size. A brown-black pigmented mass inhibited (internal) inspection and examination of the middle and rear part of the eye. Special emphasis is laid on the insecure behaviour of the foal. A connection with an iridocyclochoroiditis, which was treated in the mare about a year ago, and the pathological changes in the eye of the foal is not evident.

[Emotional experience and subjective illness perception in chronic aphasia--a comparison of patients with their family members]. / Emotionales Erleben und subjektive Krankheitswahrnehmung bei chronischer Aphasie--ein Vergleich zwischen Patienten und deren Familienangehörigen.

The results of a pilot study are demonstrated. 10 chronically aphasic patients and their relatives were interviewed by a semi-structured questionnaire. Main themes are the subjective perception and the emotional experience of psychosocial changes, psychological disturbances, communicative and neuropsychological impairments, as well as the perception of restraints caused by symptoms of disease. Methodological difficulties which arise from linguistic and communicative restrictions of aphasic patients are discussed. Data show clear emotional restraints of aphasics and their relatives in occupational, social, psychological and communicative areas. The results indicate independence of subjectively perceived alteration and its emotional experience. The differences of the emotional experience of aphasics and their relatives are discussed. While aphasics are more burdened by restrictions of independence, their relatives gave more importance to emotional changes and familiar problems.