Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Immunohistochemical Detection of Sialyl LeX Antigen on Mucosal Langerhans Cells of Human Oral Mucosa following Neuraminidase Pretreatment

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostain-ing with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le^X) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le^y. Le^a, Le^b) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le^X antigen).     

Cohort study of predictive value of urinary albumin excretion for atherosclerotic vascular disease in patients with insulin dependent diabetes.

OBJECTIVE: To examine whether slightly elevated urinary albumin excretion precedes development of atherosclerotic vascular disease in patients with insulin dependent diabetes independently of conventional atherogenic risk factors and of diabetic nephropathy. DESIGN: Cohort study with 11 year follow up. SETTING: Diabetes centre in Denmark. SUBJECTS: 259 patients aged 19-51 with insulin dependent diabetes of 6-34 years'' duration and without atherosclerotic vascular disease or diabetic nephropathy at baseline. MAIN OUTCOME MEASURES: Baseline variables: urinary albumin excretion, blood pressure, smoking habits, and serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor. End point: atherosclerotic vascular disease assessed by death certificates, mailed questionnaires, and hospital records. RESULTS: Thirty patients developed atherosclerotic vascular disease during follow up of 2457 person year. Elevated urinary albumin excretion was significantly predictive of atherosclerotic vascular disease (hazard ratio 1.06 (95% confidence interval 1.02 to 1.18) per 5 mg increase in 24 hour urinary albumin excretion, P = 0.002). Predictive effect was independent of age; sex; blood pressure; smoking; serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor; level of haemoglobin A(lc); insulin dose, duration of diabetes, and diabetic nephropathy (hazard ratio 1.04 (1.01 to 1.08) per 5 mg increase     

DNA immunization: Effects of vehicle and route of administration on the induction of protective antiviral immunity

Abstract The effectiveness of DNA immunization has been demonstrated in several model systems, usually following intramuscular injection of DNA in saline, or topical administration to the skin. In this study we have compared DNA delivered by three routes (intramuscular, intravenous, and intraperitoneal) and, for each route, in two vehicles (cationic liposome complex and pH sensitive liposome). These two lipid vehicles were evaluated because they are frequently used in gene therapy studies, but their immunogenicity has not been extensively studied. Each of these six combinations has been evaluated not only by assay of marker gene expression in a variety of tissues, but also by measurement of biologically-relevant parameters of immunity induction of antibodies, cytotoxic T lymphocytes, and protection against viral challenge. By both criteria (marker gene expression and induced immunity), the outcomes vary markedly among the six combinations. The combination leading to maximal marker gene expression (DNA with cationic lipid, administered i.v.) also induces detectable antibodies and CTL, and is the only one of the six combinations to induce immune responses comparable to those seen following i.m. injection of DNA in saline. However, marker gene expression can be detected in other combinations in the absence of induced immunity thus the value of marker gene expression in predicting the protection induced by a microbial antigen is questionable suggesting that, when evaluating various promoter constructs, marker gene expression may not adequately replace the direct measurement of biological outcomes.     

Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama