Altered degradation of circulating nucleic acids and oligonucleotides in diabetic patients.

Foreign, infection-associated or endogenously generated circulating nucleotide motifs may represent the critical determinants for the activation of the Toll-like receptors (TLRs), leading to immune stimulation and cytokine secretion. The importance of circulating nucleases is to destroy nucleic acids and oligonucleotides in the blood stream and during cell entry. Patients with juvenile insulin-dependent diabetes, adult patients with insulin-dependent diabetes and adult patients with type 2 diabetes were allocated to the study, together with the age-matched control subjects. Plasma RNase and nuclease activity were examined, in relation to different substrates-TLRs response modifiers, and circulating RNA and oligonucleotides were isolated. The fall in enzyme activity in plasma was obtained for rRNA, poly(C), poly(U), poly(I:C), poly(A:U) and CpG, especially in juvenile diabetics. In order to test the non-enzymatic glycation, commercial RNase (E.C.3.1.27.5) and control plasma samples were incubated with increasing glucose concentrations (5, 10, 20 and 50 mmol/l). The fall of enzyme activity was expressed more significantly in control plasma samples than for the commercial enzyme. Total amount of purified plasma RNA and oligonucleotides was significantly higher in diabetic patients, especially in juvenile diabetics. The increase in the concentration of nucleotides corresponded to the peak absorbance at 270 nm, similar to polyC. The electrophoretic bands shared similar characteristics between controls and each type of diabetic patients, except that the bands were more expressed in diabetic patients. Decreased RNase activity and related increase of circulating oligonucleotides may favor the increase of nucleic acid "danger motifs", leading to TLRs activation.

RNAs in the sera of Persian Gulf War veterans have segments homologous to chromosome 22q11.2.

Reverse transcriptase PCR (RT-PCR) was used for polyribonucleotide assays with sera from deployed Persian Gulf War veterans with the Gulf War Syndrome and a cohort of nonmilitary controls. Sera from veterans contained polyribonucleotides (amplicons) that were obtained by RT-PCR and that ranged in size from 200 to ca. 2,000 bp. Sera from controls did not contain amplicons larger than 450 bp. DNA sequences were derived from two amplicons unique to veterans. These amplicons, which were 414 and 759 nucleotides, were unrelated to each other or to any sequence in gene bank databases. The amplicons contained short segments that were homologous to regions of chromosome 22q11.2, an antigen-responsive hot spot for genetic rearrangements. Many of these short amplicon segments occurred near, between, or in chromosome 22q11.2 Alu sequences. These results suggest that genetic alterations in the 22q11.2 region, possibly induced by exposures to environmental genotoxins during the Persian Gulf War, may have played a role in the pathogenesis of the Gulf War Syndrome. However, the data did not exclude the possibility that other chromosomes also may have been involved. Nonetheless, the detection of polyribonucleotides such as those reported here may have application to the laboratory diagnosis of chronic diseases that have a multifactorial etiology.

[Resistance of natural and synthetic polyribonucleotide inducers of interferon to human blood ribonucleases]. / Ustoichivost' prirodnykh i sinteticheskikh poliribonukleotidnykh induktorov interferona k ribonukleazam chelovecheskoi krovi.

The resistance of polyribonucleotide inductors of interferon to blood ribonucleases was studied. Blood resistance of larifan and ridostin in the free and shielded state as well as that of the complexes of poly(I)-poly(C) and poly(G)-poly(C) were also investigated. A protective action of polylysine against the inductors was detected which, in case it had no effect on the biological activity of the drugs, could provide its recommendation as a compound for shielding the inductors.

An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter.

The promoter region of the chicken adult beta-globin gene contains a sequence of 16 deoxyguanosine residues located at a nucleosome boundary in tissues where the gene is inactive. In definitive erythrocytes that express the beta-globin gene, the nucleosome is displaced, the G-string and adjacent sequences are occupied by sequence-specific DNA-binding proteins, and a nuclease hypersensitive domain is generated in this region. To gain insight into the role of the G-string in this series of events, we have examined the proteins that bind to it. Using the gel mobility shift assay and a monoclonal antibody that blocks specific binding to the G-string, we have identified a specific protein, BGP1, that is found only in chicken erythroid cells and appears at the same time, or shortly before, the changes in chromatin structure. The antibody interacts strongly with BGP1 and cross-reacts weakly with Sp1. Although both BGP1 and Sp1 require Zn2+ for their DNA-binding activity, these proteins differ in their binding-site specificities, chromatographic properties, and molecular weights. In contrast to Sp1, which is found in a wide variety of cell types, BGP1 is restricted to erythrocytes and is most abundant in definitive erythrocytes. Thus, its presence corresponds to the tissue- and stage-specific occupancy of the G-string in vivo.

Nucleic acid hybridization in plasma: method for the quantitation of poly(I).poly(C12, U) in plasma of cancer patients.

Using a new method for the direct measurement of the double-stranded RNA (dsRNA) molecule poly(I).poly(C12, U) in plasma, levels of 100 X 10(-9) g of drug were routinely quantified. The samples were digested by proteinase K in a buffered solution containing 0.1% of Brij-35 and deoxycholate detergents. The digestions were terminated after 1 h by the addition of Brij-58 and boiling saturated NaI (1.67 g/ml). Serially diluted samples were filtered onto nitrocellulose and the filters washed and hybridized. Levels of the hybridized-radioactive probe, synthesized de novo in an RNA dependent DNA transcription system, were determined by liquid scintillation spectrophotometry and quantified by comparison to a standard curve. The efficiency of hybridization declined when the plasma concentration in the reaction fell below 1.0 mg/ml. Incubation and denaturation temperatures significantly altered the amount of radioactive probe hybridized; results varied in the extent of hybridization and in the concentration range of dsRNA showing a linear response. Elevated temperature during proteinase K digestion showed reduced hybridization efficiencies: 100% at 25, 80% at 37, 35% at 45, and 25% at 55 degrees C. Incubation at elevated temperatures, prior to the addition of NaI, caused a decline in the amount of radioactivity hybridized, but did not have an effect during hybridization.

Clinical studies with ampligen (mismatched double-stranded RNA).

Results of an ongoing clinical study of a mismatched double-stranded (ds) RNA, termed Ampligen, in patients with metastatic cancer are described. In a pilot study of Ampligen (lot 1) involving mostly hematologic malignancies, patients received cumulative doses up to approximately 450 mg without untoward effects. Evidence of biologic/antitumor effects was observed (3/5 patients) by monitoring tumor-specific markers or tumor cell morphology. In patients with solid tumors receiving lot 2, Ampligen cumulative doses over 4 g were well tolerated. The drug was given by intravenous infusion (10-80 mg/infusion, twice weekly), in some instances for more than 1 year, without clinically significant side effects. Specifically, no evidence of hematologic, liver, or renal toxicity, which was previously noted with other dsRNAs, was observed. Side effects consisted of occasional mild fatigue or flu-like symptoms. Fever, when encountered, was transient and low grade (less than 100.5 degrees F). Importantly, an analysis of patient sera for dsRNA antibodies revealed that no patient had evidence of specific antibodies directed against Ampligen. Other dsRNAs cause up to a 60% incidence of antibody formation. Additionally, a novel method was developed to monitor Ampligen blood levels. In a survey of seven patients, Ampligen had a mean plasma half-life of 23 min. Ampligen administration can also result in activation of both natural killer (NK) cells and a lymphocyte, interferon-associated, intracellular enzyme. Dose-dependent antitumor effects were seen in several solid tumors; in doses of 10-40 mg, 3/9 patients showed stable disease for up to 1 year. At the 80-mg dose level, 2/5 patients showed tumor regressions (mixed and partial responses).(ABSTRACT TRUNCATED AT 250 WORDS)

[The value of poly-C-specific serum ribonuclease and CEA in the diagnosis of pancreatic carcinoma (author's transl)]. / Zur Anwendbarkeit der Poly-C-spezifischen Serum Ribonuklease und des CEA in der Diagnose des Pankreaskarzinoms.

The possible role of poly(C)RNase serum activity and CEA serum level for early detection and differentiation of pancreatic carcinoma and its specificity and valuability were critically analyzed: Serum RNase (median, min-max) with polycytidin as substrate was determined in 13 "normal" patients (14.6 E/ml, 4.3--29.8 E/ml), 16 patients with pancreatic cancer (T3 or metastases) (17.6 E/ml, 6--49-9 E/ml), 15 patients with chronic pancreatitis (9.5 E/ml, 4.9--26.5 E/ml), 7 patients with acute pancreatitis (14.2 E/ml, 5.5--67.3 ng/ml), and 13 patients with other types of malignomas (15 E/ml, 4.3--42.5 E/ml). Serum CEA level was evaluated in 18 "normal" patients (1.15 ng/ml, 0--4.3 ng/ml), 12 patients with pancreatic carcinoma (T3 or metastases) (6.5 mg/ml, 2--456.5 ng/ml), 13 patients with chronic pancreatitis (2.3 ng/ml, 0--8.5 ng/ml), 8 patients with acute pancreatitis (2.7 ng/ml, 0.1--4.6 ng/ml) and 5 patients without operative verification of suspected pancreatic carcinoma (0.9 ng/ml, 0--1.7 ng/ml). The serum RNase activity in pancreatic cancer patients did not show any significant increase in comparison to the other groups, and these patients could not be distinguished from those with the other diseases when excluding other factors influencing serum RNase level such as: Renal insufficiency, nutrition, age, sex. Their CEA level was significantly higher in comparison to the other groups (p less than 0.05). Using 2.5 ng/ml as the limit, the sensitivity was found to be 80% (10/12 of pancreatic carcinomas positive) and the specificity being 70.5% (31/44 of other groups without malignant diseases negative). The presented study and data in the literature show that poly (C) RNase measurement is not useful in early detection of pancreatic carcinoma, but the CEA test could be helpful in the differential diagnosis of pancreatic diseases due to its specificity (70.5%) and seems to be valuable in detection of residual and in monitoring for recurrent pancreatic carcinoma in view of its sensitivity and correlation with the stage of cancer.