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Biomedical subjects

R Reddy

Publications and source records attributed to R Reddy.

At least 19 recordsLinked to original sources

Characterization of antibodies against methyl-pppN cap structure: plant U3 small nucleolar RNA is recognized by these antibodies.

In eukaryotes, many small nuclear RNAs contain either a trimethylguanosine cap structure of a gamma-monomethyl (me) cap structure. Previously, we reported the characterization of anti-mepppG antibodies which recognize methyl-capped RNAs with G as the initiation nucleotide. We report here the preparation of antibodies against mepppN cap structure. Anti-mepppN antibodies recognized only mepppN from a mixture of mepppN and pppN and immunoprecipitated mepppA-capped U3 small nucleolar RNA from a mixture of cowpea cell RNAs. These anti-mepppN antibodies recognized methylated nucleoside triphosphates (mepppA, mepppC, mepppG and mepppU) with nearly equal efficiency; however, these antibodies did not recognize methyl phosphate or methylated mononucleotides. These antibodies will be useful in the identification and characterization of all methyl-capped RNAs no matter which is the initiation nucleotide.

Antibodies, Antinuclear

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

Animals

Compilation of small RNA sequences.

This is an update containing small RNA sequences published during 1991. Approximately two hundred small RNA sequences are available in this and earlier compilations. The hard copy print out of this set will be available directly from us (inquiries should be addressed to R. Reddy). These files are also available on GenBank computer. Sequences from various sources covered in earlier compilations (see Reddy, R. Nucl. Acids Res. 16:r71; Reddy, R. and Gupta, S. Nucl Acids Res. 1990 Supplement, 18:2231 and 1991 Supplement, 19:2073) are not included in this update but are listed below.

Animals

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.

Animals

Soluble proteins delivered to dendritic cells via pH-sensitive liposomes induce primary cytotoxic T lymphocyte responses in vitro.

Effective immunity to many infectious agents, particularly viruses, requires a CD8+ cytotoxic T lymphocyte (CTL) response. Understanding how to achieve CTL induction with soluble proteins is important for vaccine development since such antigens are usually not processed appropriately to induce CTL. In the present report, we have demonstrated that a potent primary CTL response against a soluble protein can be achieved by delivering antigen in pH-sensitive liposomes to dendritic cells (DC) either in vivo or in vitro. Since the pH-sensitive liposome delivery system is efficient and easy to use, the approach promises to be valuable both in the study of basic mechanisms in antigen processing, and as a practical means of immunization.

Animals

Prevalence of antibodies to hepatitis C virus among patients with cryptogenic chronic hepatitis and cirrhosis.

Many cases of chronic hepatitis and cirrhosis cannot be attributed to a known cause and are collectively referred to as cryptogenic chronic liver disease. We have evaluated the role of the hepatitis C virus in the pathogenesis of this condition in a retrospective serum analysis for antibody to hepatitis C virus in 129 patients with cryptogenic liver disease. Other causes of chronic hepatitis and cirrhosis were ruled out by clinical, serum biochemical and serological techniques. All 129 patients were HBcAg negative, but 28 (22%) had antibody to HBcAg. Sera were tested by radioimmunoassays using recombinant peptides for antibodies to nonstructural (C100-3 and C33c) and structural regions (C22) of HCV. Among the 129 patients, 61 (47%) had antibody to C100-3, 76 (59%) had antibody to C33c and 74 (57%) had antibody to C22. Seventy-nine (61%) were reactive with at least one and 76 (59%) were reactive with at least two HCV peptides (this is the criterion used for hepatitis C virus antibody reactivity). A proportion of patients with chronic hepatitis and cirrhosis (55 of 91; 60%) similar to that of patients without cirrhosis (21 of 38; 55%) had hepatitis C virus antibody. No significant clinical, serum biochemical or histological differences were noted between the group of patients with hepatitis C virus antibody and those without this antibody reactivity. Thus more than half the patients with cryptogenic chronic liver disease had hepatitis C virus antibody, suggesting that chronic HCV infection plays a major role in the origin of cryptogenic chronic hepatitis and cirrhosis.

Adult

Sensitivity of in vivo MRS of the N-delta proton in proximal histidine of deoxymyoglobin.

The sensitivity of in vivo MRS of the N-delta proton of the proximal histidine of deoxymyoglobin in human skeletal muscles is discussed. Longitudinal relaxation time T1 of this deoxymyoglobin signal was measured in cuffed human forearms at 1.5 T and found to be 9.9 ms. Deoxymyoglobin spectra can be obtained from a forearm in seconds. The detection sensitivity of deoxymyoglobin in fully ischemic skeletal muscles and that of 31P MRS of PCr in normal resting muscles are compared.

Adult

Methylated cap structures in eukaryotic RNAs: structure, synthesis and functions.

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.

Animals

Influence of pressure supports on joint range of motion.

Burn patients often complain of restricted mobility following application of elasticized nylon anti-burn-scar supports. This study was designed to analyse the influence of this type of pressure garment on joint range of motion (ROM). Data were compiled from 80 burn-affected joints of 17 burn patients placed in a support for the first time. Joint ROM was measured directly prior to, and again immediately following, the donning of the pressure support. ROM was measured using standard goniometric techniques. Range of motion increased in 26 joints (32.5 per cent), decreased in 26 joints (32.5 per cent) and showed no change in 28 joints (35 per cent) after application of the pressure garment. There were no correlations between ROM results and age, time postburn and per cent TBSA in this population. Patients were also asked if movement felt any different after the support was applied. Their varied comments, such as movement felt easier or more difficult, were consistent with the actual ROM data. It appears that there is no predictably detrimental change in joint ROM due to the application of pressure garments.

Adult

Phase I study of high dose 5-fluorouracil and high dose Leucovorin with low dose phosphonacetyl-L-aspartic acid in patients with advanced malignancies.

Twenty-eight patients with refractory advanced malignancies were treated with a 24 hr infusion of 5-fluorouracil (5-FU), Leucovorin (LV), and N-(phosphonacetyl)-L-aspartic acid (PALA) weekly. Twenty-seven patients were evaluable for the assessment of toxicity and anti-tumor activity. PALA was administered as intravenous bolus over 15 min at a fixed dose, 250 mg/m2 24 hr before the start of 5-FU and LV infusions. 5-FU was initially administered at 750 mg/m2 and was incrementally increased to 2600 mg/m2. LV was administered in a fixed dose of 500 mg/m2 concurrently with 5-FU over a 24-hr period. The course was repeated weekly. Diarrhea, stomatitis, nausea, and vomiting were among dose-limiting toxic effects. Other toxicities observed were hand-foot syndrome, hair loss of scalp/eyelashes, overall weakness, rhinitis, and chemical conjunctivitis. Maximum tolerated dose (MTD) of 5-FU in this combination and schedule was 2600 mg/m2. Seven of 14 patients treated at 2600 mg/m2 were able to tolerate the chemotherapy on a weekly basis without interruption. The other seven patients required dose de-escalation, a majority of whom contained 5-FU at a dose of 2100 mg/m2. Twenty-three of 27 patients had been previously treated. Eight patients achieved a partial response, all of whom were previously treated, except three patients. A complete response was observed in a patient with pancreatic carcinoma, previously untreated. Overall response rate for the patients who were treated at the 5-FU dose of 2100 mg/m2 or 2600 mg/m2 is 9 of 18 patients (50%).

Adult

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.

Adenosine Triphosphate

Comparison of negative symptoms in schizophrenic and poor outcome bipolar patients.

Using the Scale for the Assessment of Negative Symptoms (SANS), affective blunting, alogia, and attentional impairment were assessed in 30 manic patients with chronic impairment of inter-episode instrumental functioning and 85 chronic schizophrenic patients. The schizophrenic patients had markedly higher ratings on all three negative symptom dimensions. When negative symptoms were examined categorically, no manic patient was rated to show prominent affective flattening or alogia. This relative specificity may not apply to attentional impairment which was rated as prominent in 17% of the manic patients and in 55% of the schizophrenic patients.

Activities of Daily Living

Contrast nephropathy in patients with impaired renal function: high versus low osmolar media.

Prescription of low osmolar contrast to prevent nephrotoxicity in subjects with pre-existing renal impairment is costly and has not been clearly shown to be effective. We entered 249 subjects with a pre-contrast serum creatinine greater than 120 mumol/liter (1.35 mg/dl) having cardiac catheterization or intravenous contrast into a randomized controlled trial comparing high and low osmolar contrast. The outcome assessed was a rise in serum creatinine repeated 48 to 72 hours after contrast. A further 117 patients entered the non-randomized prospective arm of the study. In the randomized study the serum creatinine rose by at least 25% after contrast in 8 of 117 (6.8%) given high and in 5 of 132 (3.8%) given low osmolar contrast (P greater than 0.05, one-tailed 95% confidence interval for the difference 3 to 7.8%). More severe renal failure (greater than 50% increase in serum creatinine) after contrast was uncommon (3.4% with high and 1.5% with low osmolar contrast). A rise in serum creatinine after contrast was significantly associated with the severity of the pre-contrast renal impairment and the presence of diabetes mellitus, but not with type of contrast. Diabetics with a serum creatinine greater than 200 mumol/liter (2.25 mg/dl) pre-contrast had a highest risk of deterioration in renal function after contrast. We conclude that in patients with pre-existing renal impairment the incidence of contrast nephropathy was not significantly different comparing high osmolar and nonionic contrast. The potential benefit of nonionic contrast in moderate renal impairment is likely to be small, but trials in diabetics with severe renal impairment should be undertaken urgently.

Aged

Liposomes as antigen delivery systems in viral immunity.

Since their first description, liposomes have been put to a wide variety of uses. Encapsulation or incorporation of antigens into liposomes markedly enhances the immunogenicity of the antigen. The type of immune response elicited by the liposome is found to depend on their chemical and structural properties. Immunization with viral glycoproteins encapsulated in liposomes has resulted in enhancement of the humoral response seen as a rise in the serum antibody levels which is several fold higher than that elicited by free antigen alone. Furthermore, liposome encapsulation of peptide antigens which are poor immunogens by themselves not only increases the immunogenicity of the peptide but also play an important role in delivery. The adjuvant effect afforded by liposomes can be further enhanced by the concomitant encapsulation of adjuvants like lipid A or muramyl tripeptide-phosphatidylethanolamine. Formation of liposomes with special characteristics such as pH sensitivity has resulted in the use of liposomes to deliver soluble antigen to the cytosol where it can undergo class I pathway of processing and presentation. Therefore liposomes could provide valuable tools to further understand the pathways of antigen processing and the requirements for induction of cell mediated immunity.

Animals

5' flanking sequences of human MRP/7-2 RNA gene are required and sufficient for the transcription by RNA polymerase III.

Human mitochondrial RNA processing (MRP) RNA is a 270 nucleotide-long small RNA found as ribonucleoprotein particles. In this study, we isolated four human genomic clones with homology to human MRP RNA. Two of these clones contained one copy each of the real gene coding for human MRP RNA; the other two clones represented a processed psuedogene. The Southern blot with the genomic DNA showed that the haploid human genome contains one copy of real gene and a few pseudogenes for MRP/7-2 RNA. The human MRP RNA is synthesized by RNA polymerase III and the 5' flanking sequences -84 to 1 of MRP RNA gene, containing TATA and PSE-like elements, are required and sufficient for transcription in vitro.

Animals