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.     

Antibody mimicking the action of RAS proteins on yeast adenylyl cyclase: implication for RAS-effector interaction.

Polyclonal antisera were raised against various subregions of Saccharomyces cerevisiae adenylyl cyclase in order to examine the molecular mechanism of interaction between adenylyl cyclase and RAS proteins. One of the antisera was found to activate adenylyl cyclase to an extent comparable to that activated by saturating amounts of yeast RAS2 protein produced in Escherichia coli. The stimulatory effect of this antiserum was shown to be additive with RAS2 protein when both antisera and RAS2 protein were present at low concentrations. At saturating amounts of RAS2 protein, the antisera did not exhibit additional stimulatory effects, suggesting that the actions of RAS2 protein and the antisera are complementary with each other. The antigenic determinant for the antibody involved in the activation was mapped to a 14-amino-acid segment, 1452-NSVDNGADVANLSY-1465, located between the leucine-rich repeats and the catalytic domain of adenylyl cyclase. Certain missense mutations affecting this 14-amino acid segment significantly reduced the response of adenylyl cyclase to both activating antibody and RAS proteins. These results suggest that this segment of adenylyl cyclase is intimately involved in the mechanism by which RAS proteins activate this downstream effector.     

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     

Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression.

We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation. In normal organs it is preferentially expressed in both fetal and adult lymphohematopoietic tissues. A Spa-1-encoded protein of 68 kDa is localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rap1 GTPase-activating protein (GAP), and a fusion protein of this domain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ras or Rho in vitro. Unlike the human Rap1 GAP, however, SpanN also exhibited GAP activity for Ran, so far the only known Ras-related GTPase in the nucleus. In the presence of serum, stable Spa-1 cDNA transfectants of NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NIH/Spa-1 cells were serum starved to be arrested in the G1/G0 phase of the cell cycle, however, they, unlike the control cells, exhibited progressive Spa-1 p68 accumulation, and following the addition of serum they showed cell death resembling mitotic catastrophes of the S phase during cell cycle progression. The results indicate that the novel nuclear protein Spa-1, with a potentially active Ran GAP domain, severely hampers the mitogen-induced cell cycle progression when abnormally and/or prematurely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with the Ran/RCC-1 system, which is involved in the coordinated nuclear functions, including cell division.