Specificity of hammerhead ribozyme cleavage

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells

The DD genotype is a polymorphism of the angiotensin-converting enzyme (ACE) gene, and is associated with a significantly increased risk of myocardial infarction. As endothelial dysfunction is an important event in both early atherogenesis and late atherosclerosis, we hypothesised that the adverse effect associated with the ACE/DD genotype might be mediated via endothelial damage. Using high resolution ultrasound, we studied the brachial arteries of 184 subjects aged 15-73 (mean 38 +/- 14) years, who were all normotensive, non-diabetic lifelong non-smokers. Arterial diameter was measured at rest, during reactive hyperaemia (with flow increase causing endothelium-dependent dilation) and after sublingual glyceryl trinitrate (GTN, an endothelium-independent vasodilator). The ACE genotype was determined in each case by DNA amplification; 49/184(27%) had DD, 89 (48%) had ID and 46 (25%) had II genotype. Flow-mediated dilation (FMD) was 8.5% +/- 3.9% in the DD, 7.8% +/- 4.1% in the ID and 7.8% +/- 4.1% in the II subjects (P = NS). GTN-induced dilation was also similar in the 3 groups. On multivariate analysis, endothelium-dependent dilation was inversely related to age (r = -0.33, P < 0.001), vessel size (r = -0.41, P < 0.001) but not ACE genotype (r = 0.002, P = 0.97). The ACE genotype is unrelated to endothelium-dependent dilation in the systemic arteries of clinically well adults. This suggests that the risk associated with this polymorphism may be mediated by other mechanisms.     

Substrate specificity of the ultraviolet-endonuclease from Micrococcus luteus. Endonucleolytic cleavage of depurinated DNA

The ultraviolet-endonuclease isolated from Micrococcul luteus, specific for pyrimidine dimers, is able to attack not only ultraviolet-irradiated DNA (leading to 3'OH-5'PO4 single-strand breaks) but also superhelical covalently-closed circular DNA of phage lambda damaged by heating at 70 degrees C, pH 5.93. The number of endonuclease-sensitive defects in the DNA corresponds to the number of alkalilabile bonds (apurinic sites) induced by heating. Competition between ultraviolet-induced lesions and apurinic sites for ultraviolet-endonuclease is demonstrated; the affinity of the enzyme for pyrimidine dimers is about three times that for apurinic sites. Both activities of the ultraviolet-endonuclease are inactivated at 50 degrees C at the same rate. The ultraviolet-endonuclease is able to reduce the infectious activity of depurinated lambda DNA towards Ca2+-treated uvr+ and uvr A Escherichia coli cells. It is concluded that both pyrimidine dimers and apurinic sites can be recognized by one and the same enzyme (the ultraviolet-endonuclease).     

Crystal structure of a hepatitis delta virus ribozyme

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 A resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5'-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.     

Monitoring retroviral RNA dimerization in vivo via hammerhead ribozyme cleavage

We have used a strategy for colocalization of Psi (Psi)-tethered ribozymes and targets to demonstrate that Psi sequences are capable of specific interaction in the cytoplasm of both packaging and nonpackaging cells. These results indicate that current in vitro dimerization models may have in vivo counterparts. The methodology used may be applied to further genetic analyses on Psi domain interactions in vivo.     

Substrate specificity of human prolyl-4-hydroxylase

BACKGROUND: The mechanism responsible for the forward blood flow associated with external chest compression is still controversial. Evidence for both blood flow caused by direct cardiac compression and blood flow generated by a general increase in intrathoracic pressure has been found in experimental as well as clinical studies. No data are available concerning the mechanism causing forward blood flow in hypothermic patients undergoing cardiopulmonary resuscitation. Therefore, echocardiographic findings during external chest compression in seven hypothermic arrest victims are reported. METHODS: All transesophageal echocardiographic studies performed at the Anaesthesia department between 1994 and 1997 were reviewed and seven hypothermic patients with transesophageal echocardiography performed during cardiopulmonary resuscitation were identified. RESULTS: An open mitral valve or a circumferential reduction in aortic diameter during the compression phase was found in four of seven patients, indicating that primarily an increase in intrathoracic pressure (thoracic pump mechanism) generated forward blood flow. In three patients, mitral valve closure during external chest compression indicated that direct cardiac compression (cardiac pump mechanism) contributed to forward blood flow. Two patients studied during active compression-decompression cardiopulmonary resuscitation demonstrated enhanced right ventricular filling and aortic valve opening during active decompression of the thorax. CONCLUSIONS: In contrast to normothermic arrest victims, an open mitral valve during external chest compression is a common finding during hypothermia, indicating that thoracic pump mechanism is important for forward blood flow during cardiopulmonary resuscitation in hypothermic arrest victims. Aortic valve opening in two hypothermic arrest victims suggests forward blood flow also during active decompression of the thorax with the Cardiopump.     

Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb

Terbium(III) [Tb(III)] was shown to inhibit the hammerhead ribozyme by competing with a single magnesium(II) ion. X-ray crystallography revealed that the Tb(III) ion binds to a site adjacent to an essential guanosine in the catalytic core of the ribozyme, approximately 10 angstroms from the cleavage site. Synthetic modifications near this binding site yielded an RNA substrate that was resistant to Tb(III) binding and capable of being cleaved, even in the presence of up to 20 micromolar Tb(III). It is suggested that the magnesium(II) ion thought to bind at this site may act as a switch, affecting the conformational changes required to achieve the transition state.     

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH.     

The enigmatic specificity of gamma delta T cells

In most scientific investigations, the study of mechanism follows the study of function. For example, alpha beta T cells were shown to be important mediators of immunity before the interaction between the T cell receptor (TCR) and peptide-MHC complexes was understood. However, sometimes the study of function follows from the study of mechanism. Research of gamma delta T cell receptors falls into this category. The gamma chain of the TCR was first cloned in 1984, which then led to the discovery of gamma delta T cells in 1985. Since then, research has focused on understanding ligands of the gamma delta TCR with the hope of better understanding the function of gamma delta T cells. An initial assumption was that gamma delta T cells, like alpha beta T cells, recognize peptides bound to MHC molecules; however, recent data indicate that gamma delta T cells are not biased towards MHC recognition in the same way as alpha beta T cells. Although there are intriguing new insights, the specificity and function of gamma delta T cells remains a mystery.     

Substrate specificity determinants in the farnesyltransferase beta-subunit

Protein prenyltransferases catalyze the covalent attachment of isoprenoid lipids (farnesyl or geranylgeranyl) to a cysteine near the C terminus of their substrates. This study explored the specificity determinants for interactions between the farnesyltransferase of Saccharomyces cerevisiae and its protein substrates. A series of substitutions at amino acid 149 of the farnesyltransferase beta-subunit were tested in combination with a series of substitutions at the C-terminal amino acid of CaaX protein substrates Ras2p and a-factor. Efficient prenylation was observed when oppositely charged amino acids were present at amino acid 149 of the yeast farnesyltransferase beta-subunit and the C-terminal amino acid of the CaaX protein substrate, but not when like charges were present at these positions. This evidence for electrostatic interaction between amino acid 149 and the C-terminal amino acid of CaaX protein substrates leads to the prediction that the C-terminal amino acid of the protein substrate binds near amino acid 149 of the yeast farnesyltransferase beta-subunit.     

Substrate specificity of mammalian prenyl protein-specific endoprotease activity

We have previously identified proteolytic activity in rat liver microsomes that cleaves an intact tripeptide, VIS, from S-farnesylated-CVIS tetrapeptide. This enzymatic activity, termed prenyl protein-specific endoprotease (PPEP) activity, has been solubilized in CHAPS and purified 5-fold. To probe the peptide recognition features of PPEP activity, 64 tripeptides [N-acetyl-C(S-farnesyl)a1a2] were prepared and tested as competitive inhibitors of PPEP activity-catalyzed hydrolysis of N-acetyl-C(S-farnesyl)VI[3H]S. It was found that PPEP activity prefers large hydrophobic residues in the a1 and a2 positions. A subset of N-acetyl-C(S-farnesyl)a1a2 peptides were prepared in radiolabeled form, and it was found that PPEP activity preferences for these substrates correlated well in most cases with the inhibition data. The exception is that R in the a1 position does not prevent binding of peptide to PPEP activity, but such peptides are poor substrates. The anionic residue D in the a2 position is not tolerated by PPEP activity. Five farnesylated radiolabeled tetrapeptides, Ac-C(F)FM[3H]L, Ac-C(F)LI[3H]L, Ac-C(F)LL[3H]L, Ac-C(F)LM[3H]L, and Ac-C(F)VI[3H]L were prepared, and PPEP activity kinetic studies revealed that they are good substrates and show comparable KM values (2.2-13.5 microM). Ac-C(F)RL[3H]S is a poor substrate. The reported peptide binding preferences of PPEP activity should be useful in designing compounds that block the C-terminal proteolysis of prenylated proteins. Nonprenylated peptides do not bind to PPEP activity, and replacement of the farnesyl group with ann-pentadecyl group modestly reduces binding. Peptide-membrane partitioning studies were used together with theoretical arguments to fully understand the substrate specificity of PPEP activity toward these compounds.     

Substrate specificity of prostate-specific antigen (PSA)

BACKGROUND: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage. RESULTS: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. CONCLUSION: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.     

A kinetic and thermodynamic analysis of cleavage site mutations in the hammerhead ribozyme

We have produced three forms of human Fas: full-length Fas, Fas with a C-terminal deletion, and a chimera between extracellular Fas and the intracellular domain of the tumor necrosis factor receptor I p55 subunit. We transfected cell lines with these constructs to compare the relative capacity of antibody agonists and the physiological Fas ligand (FasL) to stimulate death. With two agonistic antibodies, the chimera is 100- to 1000-fold more sensitive to induction of death than the full-length Fas. The C-terminal deletion mutant also shows greatly enhanced death in comparison to the wild-type receptor. In contrast, when FasL is used to trigger the Fas pathway, wild-type Fas and the deletion mutant are similarly sensitive, whereas the chimera is 100-fold less susceptible to ligand-mediated killing than Fas. This demonstrates that antibody agonists and natural ligand can stimulate different signaling pathways and emphasizes the limitations of defining physiologically important signaling pathways solely by antibody agonists.     

Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.     

Substrate specificity of peptide:N-glycosidase from Flavobacterium meningosepticum

A case of solitary infected renal cyst is presented. The clinical features, diagnosis and treatment of this disease entity are discussed and the literature reviewed. Less than thirty cases have been reported until the last decade.     

Substrate specificity of rat liver aldehyde dehydrogenase with chloroacetaldehydes

The aim of the present study was to validate dentists' periodontal reasons for extraction by comparison with the in vitro periodontal status of extracted teeth. A national systematic random sample of Norwegian dentists (n = 500) was requested to record primary, secondary and tertiary reasons for tooth extraction for a period of 2 weeks in 1988. The response rate was 70%. The extracted permanent teeth from the first 2 patients of each dentist were collected. Of the 365 teeth, 329 satisfied the criteria for assessment of periodontal attachment after staining. Using a dissecting microscope (10x), 4 to 8 linear measurements were recorded per tooth. 159 of the 329 teeth had loss of periodontal support. Of the 93 teeth for which the dentists' reason for extraction included periodontal considerations, 1% had 1-10% loss of attachment, 59% had 11-50% loss and 40% had 51-76% loss of periodontal support. There was a significant correlation between in vitro measurements of attachment loss and a ranking of teeth on a scale from 1 to 3 based on the dentists' emphasis on periodontal reasons for extraction (The Spearman Rs = 0.29, p < 0.01). The results suggest that the forceps level for removal of teeth for periodontal reasons was set at a relatively early stage of the disease process by Norwegian dentists, and that there was a weak association between attachment loss and the dentists' emphasis on periodontal reasons for extraction.     

Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits

We studied image cytometric DNA analysis of bladder tumors to evaluate malignant potentials of bladder tumors. METHODS: Thirty nine samples were obtained by TUR from 37 patients. Nuclear DNA content of all samples were measured by image cytometer and were determined ploidy pattern by DNA histogram. RESULTS: Of 39 TCC non-diploid pattern was recognized in 50% of grade 1 cases, 73% of grade 2 cases and 100% of grade 3 cases. DNA ploidy was strictly correlated with histological grading in TCC. DNA non-diploid pattern was present in 67% of papillary tumors, 87.5% of non-papillary tumors and 100% in CIS. In diploid pattern 2 of 7 cases with grade 1 and 2 of 4 cases with grade 2 recurred. In non-diploid pattern 1 of 4 cases with grade 1, 4 of 10 cases with grade 2 and 4 of 6 cases with grade 3 recurred. There was no significant correlation between diploid and non-diploid pattern in grade 1, 2, 3. CONCLUSION: Image cytometric DNA analysis may be useful in addition to the classic and prognostic parameters of stage and grade, especially in TCC. The differences between image analysis system and flow cytometric analysis for DNA measurement were discussed.